Cells, surfaces and adhesion

This thesis concerns cell adhesion to polymer surfaces with an experimental emphasis on hydrogels. The thesis begins with a review of the literature and a synthesis of recent evidence to describe the process of cell adhesion in a given situation. The importance of understanding integrin-adhesion protein interactions and adhesion protein-surface interactions is emphasised. The experimental chapters describe three areas of investigation. Firstly, in vitro cell culture techniques are used to explore a variety of surfaces including polyethylene glycol methacrylate (PEGMA) substituted hydrogels, sequence distribution modified hydrogels and worn contact lenses. Cell adhesion to PEGMA substituted gels is found to decrease with increases in polyethylene oxide chain length and correlations are made between sequence distribution and adhesion. Worn contact lenses are investigated for their cell adhesion properties in the presence of antibodies to specific adhesion proteins, demonstrating the presence of vitronectin and fibronectin on the lenses. The second experimental chapter addresses divalent cation regulation of integrin mediated cell adhesion. Several cell types and various cations are used. Zinc, previously not regarded as an important cation in the process, is found to inhibit 3T3 cell adhesion to vitronectin that is promoted by other divalent cations. The final experimental chapter concerns cell adhesion and growth on macroporous hydrogels. A variety of freeze-thaw formed porous gels are investiated and found generally to promote cell growth rate.Interpenetrating networkbased gels (IPN) are made porous by elution of dextrin particles of varying size and loading density. These materials provide the basis for synthetic cartilage. Cartilage cells (chondrocytes) plated onto the surface of the porous IPN materials maintain a rounded shape and hence phenotypic function when a critical pore size and density is achieved. In this way, a prospective implant, made porous at the perpendicular edges contacting natural cartilage can be both mechanically stabilised and encourage the maintenance of normal matrix production at the tissue interface.

The importance of the tissue transglutaminase-fibronectin heterocomplex in the RGD-independent cell adhesion and fibronectin matrix deposition

Tissue transglutaminase (TG2) has been reported as a wound response protein. Once over-expressed by cells under stress such as during wound healing or following tissue damage, TG2 can be secreted and deposited into extracellular matrix, where it forms a heterocomplex (TG-FN) with the abundant matrix protein fibronectin (FN). A further cellular response elicited after tissue damage is that of matrix remodelling leading to the release of the Arg-Gly-Asp (RGD) containing matrix fragments by matrix matelloproteinases (MMPs). These peptides are able to block the interaction between integrin cell surface receptors and ECM proteins, leading to the loss of cell adhesion and ultimately Anoikis. This study provides a mechanism for TG2, as a stress-induced matrix protein, in protecting the cells from the RGD-dependent loss of cell adhesion and rescuing the cells from Anoikis. Mouse fibroblasts were used as a major model for this study, including different types of cell surface receptor knockout mouse embryonic fibroblasts (MEFs) (such as syndecan-4, a5, ß1 or ß3 integrins). In addition specific syndecan-2 targetting siRNAs, ß1 integrin and a4ß1 integrin functional blocking antibodies, and a specific targeting peptide against a5ß1 integrin A5-1 were used to investigate the involvement of these receptors in the RGD-independent cell adhesion on TG-FN. Crucial for TG-FN to compensate the RGD-independent cell adhesion and actin cytoskeleton formation is the direct interaction between the heparan sulfate chains of syndecan-4 and TG2, which elicits the inside-out signalling of a5ß1 integrin and the intracellular activation of syndecan-2 by protein kinase C a (PKCa). By using specific inhibitors, a cell-permeable inhibiting peptide and the detection of the phosphorylation sites for protein kinases and/or the translocation of PKCa via Western blotting, the activation of PKCa, focal adhesion kinase (FAK), ERK1/2 and Rho kinase (ROCK) were confirmed as downstream signalling molecules. Importantly, this study also investigated the influence of TG-FN on matrix turnover and demonstrated that TG-FN can restore the RGD-independent FN deposition process via an a5ß1 integrin and syndecan-4/2 co-signalling pathway linked by PKCa in a transamidating-independent manner. These data provide a novel function for TG2 in wound healing and matrix turnover which is a key event in a number of both physiological and pathological processes.

The ecology of percolating filters containing a plastic filter medium in relation to their efficiency in The treatment of domestic sewage

The suitability of a new plastic supporting medium for biofiltration was tested over a three year period. Tests were carried out on the stability, surface properties, mechanical strength, and dimensions of the medium. There was no evidence to suggest that the medium was deficient in any of these respects. The specific surface (320m2m-3) and the voidage (94%) of the new medium are unlike any other used in bio-filtration and a pilot plant containing two filters was built to observe its effects on ecology and performance. Performance was estimated by chemical analysis and ecology studied by film examination and fauna counts. A system of removable sampling baskets was designed to enable samples to be obtained from two intermediate depths of filter. One of the major operating problems of percolating filters is excessive accumulation of film. The amount of film is influenced by hydraulic and organic load and each filter was run at a different loading. One was operated at 1.2m3m-3day-1 (DOD load 0.24kgm-3day-1) judged at the time to be the lowest filtration rate to offer advantages over conventional media. The other filter was operated at more than twice this loading (2.4m3m-3day-lBOD load 0.55kgm-3day-1) giving a roughly 2.5x and 6x the conventional loadings recommended for a Royal Commission effluent. The amount of film in each filter was normally low (0.05-3kgm(3 as volatile solids) and did not affect efficiency. The evidence collected during the study indicated that the ecology of the filters was normal when compared with the data obtained from the literature relating to filters with mineral media. There were indications that full ecological stability was yet to be reached and this was affecting the efficiency of the filters. The lower rate filter produced an average 87% BOD removal giving a consistent Royal Commission effluent during the summer months. The higher rate filter produced a mean 83% BOD removal but at no stage a consistent Royal Commission effluent. From the data on ecology and performance the filters resembled conventional filters rather than high rate filters.

Wear and corrosion of mechanical seals

Mechanical seals are used extensively to seal machinery such as pumps, mixers and agitators in the oil, petrochemical and chemical industries. The performance of such machinery is critically dependent on these devices. Seal failures may result in the escape of dangerous chemicals, possibly causing injury or loss of life. Seal performance is limited by the choice of face materials available. These range from cast iron and stellited stainless steel to cemented and silicon carbides. The main factors that affect seal performance are the wear and corrosion of seal faces. This research investigated the feasibility of applying surface coating/treatments to seal materials, in order to provide improved seal performance. Various surface coating/treatment methods were considered; these included electroless nickel plating, ion plating, plasma nitriding, thermal spraying and high temperature diffusion processes. The best wear resistance, as evaluated by the Pin-on-Disc wear test method, was conferred by the sprayed tungsten carbide/nickel/tungsten-chromium carbide deposit, produced by the high energy plasma spraying (Jet-Kote) process. In general, no correlation was found between hardness and wear resistance or surface finish and friction. This is due primarily to the complexity of the wear and frictional oxidation, plastic deformation, ploughing, fracture and delamination. Corrosion resistance was evaluated by Tafel extrapolation, linear polarisation and anodic potentiodynamic polarisation techniques. The best corrosion performance was exhibited by an electroless nickel/titanium nitride duplex coating due to the passivity of the titanium nitride layer in the acidified salt solution. The surface coating/treatments were ranked using a systematic method, which also considered other properties such as adhesion, internal stress and resistance to thermal cracking. The sealing behaviour of surface coated/treated seals was investigated on an industrial seal testing rig. The best sealing performances were exhibited by the Jet-Kote and electroless nickel silicon carbide composite coated seals. The failure of the electroless nickel and electroless nickel/titanium nitride duplex coated seals was due to inadequate adhesion of the deposits to the substrate. Abrasion of the seal faces was the principal wear mechanism. For operation in an environment similar to the experimental system employed (acidified salt solution) the Jet-Kote deposit appears to be the best compromise.

A biological-inspired support frame for an artificial cornea

Bilateral corneal blindness represents a quarter of the total blind, world-wide. The artificial cornea in assorted forms, was developed to replace opaque non-functional corneas and to return sight in otherwise hopeless cases that were not amenable to corneal grafts; believed to be 2% of corneal blind. Despite technological advances in materials design and tissue engineering no artificial cornea has provided absolute, long-term success. Formidable problems exist, due to a combination of unpredictable wound healing and unmanageable pathology. To have a solid guarantee of reliable success an artificial cornea must possess three attributes: an optical window to replace the opaque cornea; a strong, long term union to surrounding ocular tissue; and the ability to induce desired host responses. A unique artificial cornea possesses all three functional attributes- the Osteo-odonto-keratoprosthesis (OOKP). The OOKP has a high success rate and can survive for up to twenty years, but it is complicated both in structure and in surgical procedure; it is expensive and not universally available. The aim of this project was to develop a synthetic substitute for the OOKP, based upon key features of the tooth and bone structure. In doing so, surgical complexity and biological complications would be reduced. Analysis of the biological effectiveness of the OOKP showed that the structure of bone was the most crucial component for implant retention. An experimental semi-rigid hydroxyapatite framework was fabricated with a complex bone-like architecture, which could be fused to the optical window. The first method for making such a framework, was pressing and sintering of hydroxyapatite powders; however, it was not possible to fabricate a void architecture with the correct sizes and uniformity of pores. Ceramers were synthesised using alternative pore forming methods, providing for improved mechanical properties and stronger attachment to the plastic optical window. Naturally occurring skeletal structures closely match the structural features of all forms of natural bone. Synthetic casts were fabricated using the replamineform process, of desirable natural artifacts, such as coral and sponges. The final method of construction by-passed ceramic fabrication in favour of pre-formed coral derivatives and focused on methods for polymer infiltration, adhesion and fabrication. Prototypes were constructed and evaluated; a fully penetrative synthetic OOKP analogue was fabricated according to the dimensions of the OOKP. Fabrication of the cornea shaped OOKP synthetic analogue was also attempted.

Elastic-plastic analysis of complete building structures

In this Thesis, details of a proposed method for the elastic-plastic failure load analysis of complete building structures are given. In order to handle the problem, a computer programme in Atlas Autocode is produced. The structures consist of a number of parallel shear walls and intermediate frames connected by floor slabs. The results of an experimental investigation are given to verify the theoretical results and to demonstrate various factors that may influence the behaviour of these structures. Large full scale practical structures are also analysed by the proposed method and suggestions are made for achieving design economy as well as for extending research in various aspects of this field. The existing programme for elastic-plastic analysis of large frames is modified to allow for the effect of composite action of structural members, i.e. reinforced concrete floor slabs and the supporting steel beams. This modified programme is used to analyse some framed type structures with composite action as well as those which incorporate plates and shear walls. The results obtained are studied to ascertain the influence of composite action and other factors on the load carrying capacity of both bare frames and complete building structures. The theoretical failure load presented in this thesis does not predict the overall failure load of the structure nor does it predict the partial failure load of the shear walls and slabs but it merely predicts the partial failure load of a single frame and assumes that the loss of stiffess of such a frame renders the overall structure unusable. For most structures the analysis proposed in this thesis is likely to break down prematurely due to the failure of the slab and shear wall system and this factor must be taken into account in any future work on such structures. The experimental work reported in this thesis is acknowledged to be unsatisfactory as a verification of the limited theory proposed. In particular perspex was not found to be a suitable material for testing at high loads, micro-concrete may be more suitable.

Numerical modelling of agglomerate degradation

In the processing industries particulate materials are often in the form of powders which themselves are agglomerations of much smaller sized particles. During powder processing operations agglomerate degradation occurs primarily as a result of collisions between agglomerates and between agglomerates and the process equipment. Due to the small size of the agglomerates and the very short duration of the collisions it is currently not possible to obtain sufficiently detailed quantitative information from real experiments to provide a sound theoretically based strategy for designing particles to prevent or guarantee breakage. However, with the aid of computer simulated experiments, the micro-examination of these short duration dynamic events is made possible. This thesis presents the results of computer simulated experiments on a 2D monodisperse agglomerate in which the algorithms used to model the particle-particle interactions have been derived from contact mechanics theories and, necessarily, incorporate contact adhesion. A detailed description of the theoretical background is included in the thesis. The results of the agglomerate impact simulations show three types of behaviour depending on whether the initial impact velocity is high, moderate or low. It is demonstrated that high velocity impacts produce extensive plastic deformation which leads to subsequent shattering of the agglomerate. At moderate impact velocities semi-brittle fracture is observed and there is a threshold velocity below which the agglomerate bounces off the wall with little or no visible damage. The micromechanical processes controlling these different types of behaviour are discussed and illustrated by computer graphics. Further work is reported to demonstrate the effect of impact velocity and bond strength on the damage produced. Empirical relationships between impact velocity, bond strength and damage are presented and their relevance to attrition and comminution is discussed. The particle size distribution curves resulting from the agglomerate impacts are also provided. Computer simulated diametrical compression tests on the same agglomerate have also been carried out. Simulations were performed for different platen velocities and different bond strengths. The results show that high platen velocities produce extensive plastic deformation and crushing. Low platen velocities produce semi-brittle failure in which cracks propagate from the platens inwards towards the centre of the agglomerate. The results are compared with the results of the agglomerate impact tests in terms of work input, applied velocity and damage produced.

Biological interactions with synthetic polymers

Anchorage dependent cell culture is a useful model for investigating the interface that becomes established when a synthetic polymer is placed in contact with a biological system. The primary aim of this interdisciplinary study was to systematically investigate a number of properties that were already considered to have an influence on cell behaviour and thereby establish the extent of their importance. It is envisaged that investigations such as these will not only further the understanding of the mechanisms that affect cell adhesion but may ultimately lead to the development of improved biomaterials. In this study, surface analysis of materials was carried out in parallel with culture studies using fibroblast cells. Polarity, in it's ability to undergo hydrogen bonding (eg with water and proteins), had an important affect on cell behaviour, although structural arrangement and crystallinity were not found to exert any marked influence. In addition, the extent of oxidation that had occurred during the process of manufacture of substrates was also important. The treatment of polystyrene with a selected series of acids and gas plasmas confirmed the importance of polarity, structural groups and surface charge and it was shown that this polymer was not unique among `hydrophobic' materials in it's inability to support cell adhesion. The individual water structuring groups within hydrogel polymers were also observed to have controlling effects on cell behaviour. An overall view of the biological response to both hydrogel and non-hydrogel materials highlighted the importance of surface oxidation, polarity, water structuring groups and surface charge. Initial steps were also taken to analyse foetal calf serum, which is widely used to supplement cell culture media. Using an array of analytical techniques, further experiments were carried out to observe any possible differences in the amounts of lipids and calcium that become deposited to tissue culture and bacteriological grade plastic under cell culture conditions.

A production planning system for plastic footwear in a seasonal market

Modern injection-moulding machinery which produces several, pairs of plastic footwear at a time brought increased production planning problems to a factory. The demand for its footwear is seasonal but the company's manning policy keeps a fairly constant production level thus determining the aggregate stock. Production planning must therefore be done within the limitations of a specified total stock. The thesis proposes a new production planning system with four subsystems. These are sales forecasting, resource planning, and two levels of production scheduling: (a) aggregate decisions concerning the 'manufacturing group' (group of products) to be produced in each machine each week, and (b) detailed decisions concerning the products within a manufacturing group to be scheduled into each mould-place. The detailed scheduling is least dependent on improvements elsewhere so the sub-systems were tackled in reverse order. The thesis concentrates on the production scheduling sub-systems which will provide most. of the benefits. The aggregate scheduling solution depends principally on the aggregate stocks of each manufacturing group and their division into 'safety stocks' (to prevent shortages) and 'freestocks' (to permit batch production). The problem is too complex for exact solution but a good heuristic solution, which has yet to be implemented, is provided by minimising graphically immediate plus expected future costs. The detailed problem splits into determining the optimal safety stocks and batch quantities given the appropriate aggregate stocks. It.is found that the optimal safety stocks are proportional to the demand. The ideal batch quantities are based on a modified, formula for the Economic Batch Quantity and the product schedule is created week by week using a priority system which schedules to minimise expected future costs. This algorithm performs almost optimally. The detailed scheduling solution was implemented and achieved the target savings for the whole project in favourable circumstances. Future plans include full implementation.