Development of a biaxial compression device for biological samples : preliminary experimental results for a closed cell foam

Biological tissues are subjected to complex loading states in vivo and in order to define constitutive equations that effectively simulate their mechanical behaviour under these loads, it is necessary to obtain data on the tissue's response to multiaxial loading. Single axis and shear testing of biological tissues is often carried out, but biaxial testing is less common. We sought to design and commission a biaxial compression testing device, capable of obtaining repeatable data for biological samples. The apparatus comprised a sealed stainless steel pressure vessel specifically designed such that a state of hydrostatic compression could be created on the test specimen while simultaneously unloading the sample along one axis with an equilibrating tensile pressure. Thus a state of equibiaxial compression was created perpendicular to the long axis of a rectangular sample. For the purpose of calibration and commissioning of the vessel, rectangular samples of closed cell ethylene vinyl acetate (EVA) foam were tested. Each sample was subjected to repeated loading, and nine separate biaxial experiments were carried out to a maximum pressure of 204 kPa (30 psi), with a relaxation time of two hours between them. Calibration testing demonstrated the force applied to the samples had a maximum error of 0.026 N (0.423% of maximum applied force). Under repeated loading, the foam sample demonstrated lower stiffness during the first load cycle. Following this cycle, an increased stiffness, repeatable response was observed with successive loading. While the experimental protocol was developed for EVA foam, preliminary results on this material suggest that this device may be capable of providing test data for biological tissue samples. The load response of the foam was characteristic of closed cell foams, with consolidation during the early loading cycles, then a repeatable load-displacement response upon repeated loading. The repeatability of the test results demonstrated the ability of the test device to provide reproducible test data and the low experimental error in the force demonstrated the reliability of the test data.

Monitoring mesenchymal stem cell cultures using image processing and pattern recognition techniques

Stem cells have attracted tremendous interest in recent times due to their promise in providing innovative new treatments for a great range of currently debilitating diseases. This is due to their potential ability to regenerate and repair damaged tissue, and hence restore lost body function, in a manner beyond the body's usual healing process. Bone marrow-derived mesenchymal stem cells or bone marrow stromal cells are one type of adult stem cells that are of particular interest. Since they are derived from a living human adult donor, they do not have the ethical issues associated with the use of human embryonic stem cells. They are also able to be taken from a patient or other donors with relative ease and then grown readily in the laboratory for clinical application. Despite the attractive properties of bone marrow stromal cells, there is presently no quick and easy way to determine the quality of a sample of such cells. Presently, a sample must be grown for weeks and subject to various time-consuming assays, under the direction of an expert cell biologist, to determine whether it will be useful. Hence there is a great need for innovative new ways to assess the quality of cell cultures for research and potential clinical application. The research presented in this thesis investigates the use of computerised image processing and pattern recognition techniques to provide a quicker and simpler method for the quality assessment of bone marrow stromal cell cultures. In particular, aim of this work is to find out whether it is possible, through the use of image processing and pattern recognition techniques, to predict the growth potential of a culture of human bone marrow stromal cells at early stages, before it is readily apparent to a human observer. With the above aim in mind, a computerised system was developed to classify the quality of bone marrow stromal cell cultures based on phase contrast microscopy images. Our system was trained and tested on mixed images of both healthy and unhealthy bone marrow stromal cell samples taken from three different patients. This system, when presented with 44 previously unseen bone marrow stromal cell culture images, outperformed human experts in the ability to correctly classify healthy and unhealthy cultures. The system correctly classified the health status of an image 88% of the time compared to an average of 72% of the time for human experts. Extensive training and testing of the system on a set of 139 normal sized images and 567 smaller image tiles showed an average performance of 86% and 85% correct classifications, respectively. The contributions of this thesis include demonstrating the applicability and potential of computerised image processing and pattern recognition techniques to the task of quality assessment of bone marrow stromal cell cultures. As part of this system, an image normalisation method has been suggested and a new segmentation algorithm has been developed for locating cell regions of irregularly shaped cells in phase contrast images. Importantly, we have validated the efficacy of both the normalisation and segmentation method, by demonstrating that both methods quantitatively improve the classification performance of subsequent pattern recognition algorithms, in discriminating between cell cultures of differing health status. We have shown that the quality of a cell culture of bone marrow stromal cells may be assessed without the need to either segment individual cells or to use time-lapse imaging. Finally, we have proposed a set of features, that when extracted from the cell regions of segmented input images, can be used to train current state of the art pattern recognition systems to predict the quality of bone marrow stromal cell cultures earlier and more consistently than human experts.

Phase III study of matrix metalloproteinase inhibitor prinomastat in non-small-cell lung cancer

Purpose: Matrix metalloproteinases (MMPs) degrade extracellular proteins and facilitate tumor growth, invasion, metastasis, and angiogenesis. This trial was undertaken to determine the effect of prinomastat, an inhibitor of selected MMPs, on the survival of patients with advanced non-small-cell lung cancer (NSCLC), when given in combination with gemcitabine-cisplatin chemotherapy. Patients and Methods: Chemotherapy-naive patients were randomly assigned to receive prinomastat 15 mg or placebo twice daily orally continuously, in combination with gemcitabine 1,250 mg/m2 days 1 and 8 plus cisplatin 75 mg/m2 day 1, every 21 days for up to six cycles. The planned sample size was 420 patients. Results: Study results at an interim analysis and lack of efficacy in another phase III trial prompted early closure of this study. There were 362 patients randomized (181 on prinomastat and 181 on placebo). One hundred thirty-four patients had stage IIIB disease with T4 primary tumor, 193 had stage IV disease, and 34 had recurrent disease (one enrolled patient was ineligible with stage IIIA disease). Overall response rates for the two treatment arms were similar (27% for prinomastat v 26% for placebo; P = .81). There was no difference in overall survival or time to progression; for prinomastat versus placebo patients, the median overall survival times were 11.5 versus 10.8 months (P = .82), 1-year survival rates were 43% v 38% (P = .45), and progression-free survival times were 6.1 v 5.5 months (P = .11), respectively. The toxicities of prinomastat were arthralgia, stiffness, and joint swelling. Treatment interruption was required in 38% of prinomastat patients and 12% of placebo patients. Conclusion: Prinomastat does not improve the outcome of chemotherapy in advanced NSCLC. © 2005 by American Society of Clinical Oncology.

Evaluation of brief dietary questions to estimate vegetable and fruit consumption – using serum carotenoids and red-cell folate

Objective: To evaluate responses to self-administered brief questions regarding consumption of vegetables and fruit by comparison with blood levels of serum carotenoids and red-cell folate. Design: A cross-sectional study in which participants reported their usual intake of fruit and vegetables in servings per day, and serum levels of five carotenoids (α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and lycopene) and red-cell folate were measured. Serum carotenoid levels were determined by high-performance liquid chromatography, and red-cell folate by an automated immunoassay system. Settings and subjects: Between October and December 2000, a sample of 1598 adults aged 25 years and over, from six randomly selected urban centres in Queensland, Australia, were examined as part of a national study conducted to determine the prevalence of diabetes and associated cardiovascular risk factors. Results: Statistically significant (P<0.01) associations with vegetable and fruit intake (categorised into groups: ≤1 serving, 2–3 servings and ≥4 servings per day) were observed for α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and red-cell folate. The mean level of these carotenoids and of red-cell folate increased with increasing frequency of reported servings of vegetables and fruit, both before and after adjusting for potential confounding factors. A significant association with lycopene was observed only for vegetable intake before adjusting for confounders. Conclusions: These data indicate that brief questions may be a simple and valuable tool for monitoring vegetable and fruit intake in this population.

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis. © 2011 Macmillan Publishers Limited. All rights reserved.

Magnetic resonance microimaging of cancer cell spheroid constructs

BACKGROUND Hydrogel-based cell cultures are excellent tools for studying physiological events occurring in the growth and proliferation of cells, including cancer cells. Diffusion magnetic resonance is a physical technique that has been widely used for the characterisation of biological systems as well as hydrogels. In this work, we applied diffusion magnetic resonance imaging (MRI) to hydrogel-based cultures of human ovarian cancer cells. METHODS Diffusion-weighted spin-echo MRI measurements were used to obtain spatially-resolved maps of apparent diffusivities for hydrogel samples with different compositions, cell loads and drug (Taxol) treatment regimes. The samples were then characterised using their diffusivity histograms, mean diffusivities and the respective standard deviations, and pairwise Mann-Whitney tests. The elastic moduli of the samples were determined using mechanical compression testing. RESULTS The mean apparent diffusivity of the hydrogels was sensitive to the polymer content, cell load and Taxol treatment. For a given sample composition, the mean apparent diffusivity and the elastic modulus of the hydrogels exhibited a negative correlation. CONCLUSIONS Diffusivity of hydrogel-based cancer cell culture constructs is sensitive to both cell proliferation and Taxol treatment. This suggests that diffusion-weighted imaging is a promising technique for non-invasive monitoring of cancer cell proliferation in hydrogel-based, cellularly-sparse 3D cell cultures. The negative correlation between mean apparent diffusivity and elastic modulus suggests that the diffusion coefficient is indicative of the average density of the physical microenvironment within the hydrogel construct.

Optimisation of in-cell accelerated solvent extraction technique for the determination of organochlorine pesticides in river sediments

Organochlorine pesticides (OCPs) are ubiquitous environmental contaminants with adverse impacts on aquatic biota, wildlife and human health even at low concentrations. However, conventional methods for their determination in river sediments are resource intensive. This paper presents an approach that is rapid and also reliable for the detection of OCPs. Accelerated Solvent Extraction (ASE) with in-cell silica gel clean-up followed by Triple Quadrupole Gas Chromatograph Mass Spectrometry (GCMS/MS) was used to recover OCPs from sediment samples. Variables such as temperature, solvent ratio, adsorbent mass and extraction cycle were evaluated and optimised for the extraction. With the exception of Aldrin, which was unaffected by any of the variables evaluated, the recovery of OCPs from sediment samples was largely influenced by solvent ratio and adsorbent mass and, to some extent, the number of cycles and temperature. The optimised conditions for OCPs extraction in sediment with good recoveries were determined to be 4 cycles, 4.5 g of silica gel, 105 ᴼC, and 4:3 v/v DCM: hexane mixture. With the exception of two compounds (α-BHC and Aldrin) whose recoveries were low (59.73 and 47.66 % respectively), the recovery of the other pesticides were in the range 85.35 – 117.97% with precision < 10 % RSD. The method developed significantly reduces sample preparation time, the amount of solvent used, matrix interference, and is highly sensitive and selective.

Effective Cell-Centred Time-Domain Maxwell's Equations Numerical Solvers

This research work analyses techniques for implementing a cell-centred finite-volume time-domain (ccFV-TD) computational methodology for the purpose of studying microwave heating. Various state-of-the-art spatial and temporal discretisation methods employed to solve Maxwell's equations on multidimensional structured grid networks are investigated, and the dispersive and dissipative errors inherent in those techniques examined. Both staggered and unstaggered grid approaches are considered. Upwind schemes using a Riemann solver and intensity vector splitting are studied and evaluated. Staggered and unstaggered Leapfrog and Runge-Kutta time integration methods are analysed in terms of phase and amplitude error to identify which method is the most accurate and efficient for simulating microwave heating processes. The implementation and migration of typical electromagnetic boundary conditions. from staggered in space to cell-centred approaches also is deliberated. In particular, an existing perfectly matched layer absorbing boundary methodology is adapted to formulate a new cell-centred boundary implementation for the ccFV-TD solvers. Finally for microwave heating purposes, a comparison of analytical and numerical results for standard case studies in rectangular waveguides allows the accuracy of the developed methods to be assessed.