Understanding intracellular processing of gene vectors will help to improve vector design in gene therapy. Chitosan nanoparticles have been previously identied as safe and non toxic gene vector. Linear chitosan oligomer (LCO) and self branched trisaccharide chitosan oligomer (SBTCO) have been shown to be able to pack DNA, balancing betweencomplexation and intracellular unpacking . However, the transfection ecacies of thesetwo chitosans diers considerably with the level of transgene expression higher for SBTCOcompared to LCO. SBTCO have been recently reported to be taken up solely by caveolaemediated endocytosis (CvME) while LCO uses both clathrin mediated endocytosis (CME)and CvME.In the present study, the CvME was studied. An immunostaining protocol for detectionof caveolin (cav) was established. Polyplexes of SBTCO seemed to trigger the formationof more caveosomes than did LCO polyplexes. SBTCO polyplexes were more localisedin the caveosome than LCO polyplexes at 3 hours incubation period. These ndingssuggested that SBTCO polyplexes delivers more DNA into the cell than LCO polyplexesand that SBTCO polyplexes are processed intracellularly solely via the CvME pathway.Likewise, it suggested LCO polyplexes have preferred intracellular processing pathwaywhich is not CvME. Collectively, these results demonstrated that SBTCO is protectedfrom the degradative endosome and might therefore be an ecient and good tool to delivertherapeutic DNA.

As the world population grows and the standard of living increases a need for better health care is an important consideration. Over the latter half of this century the use of polymeric materials within the field of medicine has grown considerably. This thesis investigates a variety of novel polymers whose applications within the medical field are both important and varied. In the cases studied, as with other polymeric materials intended for medicinal uses, the interaction with their surfaces is important, as when placed in the body the surface is the first place of contact and hence interaction with the system. Because of the importance of these surface interactions, which can elicit both beneficial and detrimental effects, this thesis is concerned with the surface chemistry and the relationship of this property to the interfacial interactions of the polymeric systems investigated. Chapter 1 will concentrate on providing a historical overview of the field of polymers, especially the applications in the biomedical field. The surface analytical techniques of surface plasmon resonance (SPR), atomic force microscopy (AFM), X-ray photoelectron spectrometry (XPS) and secondary ion mass spectrometry (SIMS) utilised in this the is to investigate the surface properties of the materials of interest will also be introduced. The demand for organ transplants far outweighs the supply of donor organs. Therefore, many patients with a variety of disease die each year. If new organs could be grown utilising the patients own cells, the supply of organs could be increased to meet the demand and these custom grown organ would not nave the problems of rejection as observed with donor ones. This is what tissue engineering aims to do. By utilising polymer matrices, to provide a scaffold for the tissue to grow in the correct configuration and a variety of growth factors and cell signalling agents to ensure the correct differentiation and function of the cells, it is hoped in the future new organs for example lung and livers will be able to be grown on demand. Chapter 3 and 4 concentrate on the in depth study of a polymer intended for such an application. Chapter 3 concentrates on determining the surface chemistry of this polymer system. XPS and SIMS are used to identify the type of chemical groups present at the surface and quantify their contribution to the overall surface layer. Chapter 4, probes the interactions, both specific and non specific with this system using a variety of complementary techniques. SPR is utilised to quantify the extent and rate of the interactions, whilst AFM in force distance mode the strength of these. AFM was also utilised to visualize the absorbed molecules on the polymer surfaces. In Chapter 5 the area of gene therapy is introduced. It is hoped that in the future, gene therapy may form the basis of a cure for inherited gene tic diseases, such as hemophilia and other conditions such as AIDS and some cancers. The main problems which need to be overcome before this aim can be realised are firstly, isolating the correct genes to cure the diseases, and secondly, delivery of these genes. Cationic polymers and cationic lipids are two of the three main areas of research being investigated to find a potential carrier system for the DNA. In Chapter 5, the effect of PEG molecular weight on the condensation of plasmid DNA into a particles by poly(L-lysine)s for gene therapy is investigated. SPR was utilised to investigated the rate and extent of DNA binding and condensation by the various polymers, and utilising AFM the effect of PEG molecular weight on the structure of the observed particles was probed. The gene therapy theme is continued in Chapter 6 where the surface interactions of a cationic lipid / DNA gene therapy complex is investigated by both AFM and SPR. There are two main aims to the studies in this chapter. Firstly, to provide an understanding of the interactions between the DNA and lipid components of the delivery system. It is hoped this will supply further information on its stability, formation and structure and secondly, to provide a knowledge of the interactions of the complex with model surfaces. This may provide an insight into the gene therapy vectors possible mode of interaction with the cell. Chapter 7, is concerned with a family of dendrimeric polymers. The family of interest are the poly(amidoamine) (PAM AM) dendrimers. These have shown potential not only as drug delivery vehicles in the field of cancer chemotherapy and gene therapy, but also in enhancing medical imaging. The investigations performed in Chapter 7 form a basis for understanding the factors effecting the PAMAM's interaction with cell membranes and hence, provide information for optimising their formulation into drug delivery vehicles. The studies undertaken utilised both AFM and SPR as well as a range of model surface. These surfaces possess differing characteristics and were ulilised in conjunction with AFM and SPR to study the effect of dendrimer size, shape, surface charge and charge density on the interaction of these molecules. The final chapter, Chapter 8 discusses the progress made towards the aims of this thesis as outlined in section 1.8 and addresses the avenues for future investigations exposed by experimental work in chapters 3 - 7. Overall it is hoped that the work described in this thesis shows that a multi technique approach, as well as the collaborations of chemists, materials engineers, biophysicist, biologists and clinicians may, in the future lead to a better understanding of the materials utilised in the medical setting and hence provide a systematic design of systems for treating specific disease conditions.

The pain experience as a result of disc degeneration disease (DDD) can be debilitating. When drug administration and physiotherapy treatment fail, surgical methods are used. These involve removal of the affected intervertebral disc IVD, followed by either decompression and fusion of the adjacent vertebral bodies or replacement of the removed IVD with an implant. Fusion is seen to be the gold standard for surgical treatment of DDD, but questions have been raised about its effectiveness in the long term due to its association with the adjacent levels disc degeneration. Disc replacement has been developed as an alternative to overcome this problem. The aim of the implant, in contrast to fusion, is the preservation of motion at the treated level. This has been said to maintain the adjacent level biomechanics and hence, prevent rapid degeneration. A novel graduated modulus polymeric total disc replacement device, Compliant Artificial Disc (CAdisc) developed by Ranier Technology Limited was studied in this project. Its design is such as to provide load-bearing capability and motion preservation at the implanted site. Through a unique manufacturing process, Precision Polyurethane Manufacturing PPM, the lower modulus ‘nucleus’ material of this device is encapsulated by the higher modulus ‘annulus’ with presence of graduated modulus in between. This project, aims to analyse the CAdisc mechanical properties and evaluate its biomechanical performance. Scanning Acoustic Microscope SAM and nano-indentation was used to analyse the CAdisc internal modulus distribution. The results show different modulus regions (the annulus, the graduated and the nucleus regions) in the CAdisc device and demonstrate the potential of the PPM process to produce consistent graduated region. It was also found that the SAM results were comparable to the nano-indentation with a significant correlation between the results. The technology in the development of the CAdisc-L (lumbar disc replacement) has been used to develop its cervical counterpart, CAdisc-C which is in its initial stage of design. Using a validated highly meshed 3D FEM of the cervical spine (C4-C7), developed from CT data, the biomechanical performance of cervical version of the CAdisc (CAdisc-C) was evaluated. The result shows the implant preserved motion at the treated level and gives a performance that preserved the biomechanics of the adjacent level compared to fusion. The study also shows that misplacement of the implant from its optimal position will not significantly affect its performance.

Due to geometrical features, it is claimed that larger femoral heads in total hip replacement (THR) are superior in achieving normal biomechanics than smaller ones; and that hip resurfacing (RSF) is superior to THR. This has not been conclusively proven. Most studies have investigated level walking, which may not be demanding enough to highlight what could be small biomechanical differences between implants. Few biomechanical studies have compared more demanding tasks and not with patients with different femoral head sizes or RSF. This thesis aimed to address these omissions by investigating level walking, stair descent and sit-to-stand (STS) biomechanics between three groups (32mm THR, 36mm THR and RSF). Twenty-six osteoarthritis patients were recruited and tested pre-operatively, then three and twelve months post-operatively. Demographic differences between groups were expected due to patient considerations for different implants, so a study was performed to determine whether level walking biomechanics alter progressively during the aging process with a group of 63 healthy participants. Three matched sub-groups were extracted from this group as controls. There was no suggestion that gait deteriorates progressively with age. Hip reconstruction, irrespective of head size, can allow patients to return to the biomechanical levels of controls during level walking. Stair descent differences remained 12 months post-operatively in cadence (p=0.042) and peak hip power generated (p<0.001) compared to controls. The 32mm group exhibited vertical ground reaction force (vGRF) asymmetry pre-operatively (p<0.001) and 3 months post-operatively (p=0.013); and impulse asymmetry (p<0.001) pre-operatively during STS. The 36mm group exhibited impulse asymmetry (p=0.05)three months post-operatively. This thesis is the first biomechanical analysis of stair descent and STS of two THR groups and a RSF group. It has demonstrated stair descent differences at 12 months post-operatively and overloading of the healthy limb in some THR patients. The latter could be problematic for the healthy limb.

Cellular transplantation, a current therapy for cardiac failure, does not consider the need for a physical support or biochemical factors required by the cardiomyocyte. The aim of this project was to establish the Extra Cellular Matrix (ECM), architectural and mechanical properties of a flexible scaffold to assist the maintenance of a cardiac cell line cultured under mechanical stimuli. Previously, mechanical stimulation has been proved to have an effect in cardiomyocytes similar to that of growth factors on other cells and promotes protein expression, differentiation and survival [1]. Poly-(1,8-octanediol-co-citric acid) [POC] is an elastomer that can be processed into scaffolds for tissue engineering. Mechanical properties of the POC were compared at different porosity, storage method and strain rate. POC, with an ultimate elongation of 60-160%, did support cardiac cell attachment when coated with fibronectin. Seeding strategies were evaluated to find optimal conditions and static seeding resulted more favourable for cell adhesion and survival than other dynamics approaches. In collaboration with the University of Leeds, cardiomyocytes were cultured in a dynamic bioreactor, Tencell, under continuous and discontinuous stretching regimes. Mechano-stimulation of cardiac constructs encouraged cell survival in the discontinuous regime and up-regulated the expression of actc1 and nppa genes regardless of the treatments. It was concluded that although mechanical stimulation had a positive effect on cell survival and gene expression, tissue formation was not promoted.

No description available.

Cell transplantation on biodegradable scaffolds is an established approach in tissue engineering to the problem of the regeneration of diseased or damaged tissues. As cells grow and organise themselves, they secrete their own extracellular matrix, while the polymer degrades into natural metabolites resulting in eventual natural tissue replacement. Polymeric materials used for these scaffolds must satisfy a number of requirements. These include defined cell-interactive properties, porosity, biodegradability, mechanical and controlled release properties. To date, scaffolds have been designed to conform to these requirements. However, the need to perform defined three-dimensional structures requires prior knowledge of the dimensions of the defect or cavity to be filled. Furthermore the general use of toxic solvents in the processing of these scaffolds prevents the incorporation of biological agents and cells during fabrication. Therefore, poor transportation of cells through the scaffolds can result in low cell seeding efficiencies. Finally such scaffolds require an invasive operation for transplantation of the material. In contrast a number of injectable materials have been proposed and investigated. The transformation from liquid pre-cursor to gel in such systems can, however, require cell harmful trigger signals such as UV exposure or pH changes. Furthermore, these injectable gels lack a porous structure preventing effective cell migration and restricting tissue formation and vascularisation tothe barrier of diffusion for signalling and nutrient molecules. The work in this thesis presents a scaffold that is both injectable and conforms to the requirements of water-insoluble porous scaffolds. This starts with the synthesis of a biotinylated poly (lactic acid)-poly (ethylene glycol) (PLA-PEG) copolymer. The polymer is degradable, protein resistant and cell interactive when used in conjunction with biotinylated cell adhesive peptides. The biotin unit tethered to the PEG-PLA also provides the polymer with self-assembling properties when used in conjunction with avidin. In contrast to alternative injectable materials, the scaffold presented in this thesis is porous. This porosity is necessary for tissue ingrowth and vascularization. Therefore, before progressing on to the manufacture of the scaffold, a systematic study of two cell types involved in vascularisation was carried out over defined pore features. These studies revealed that cell behaviour over pore features was related to cell type, cell density and pore size. This had significant implications for the injectable scaffold in development because proposed advantages were delivery of a variety of cell types, controlled porous structure, and efficient cell seeding. Microparticles were then manufactured from the PLA-PEG-biotin using a single emulsion manufacturing process. Surface Plasmon Resonance (SPR) confirmed that these microparticles would bind efficiently to avidin. The condition for optimum self-assembling of particles was then determined using aggregation studies. These studies showed that a critical quantity of avidin was required for microparticles to aggregate together. The ability to aggregate particles of different sizes leads to the potential for controlling scaffold porosity. Rheological testing showed that the scaffold's mechanical properties could be tailored to that of the tissue in which regeneration is required. The self-assembly of microparticles was also demonstrated to form complex three-dimensional scaffolds without the use of toxic solvents. Scaffolds prepared in simulated tissues maintained shape upon injection. Scaffolds were then self-assembled with cells entrapped within them. Cell viability within the self-assembling scaffolds was confirmed by Alamar Blue assays. In vivo studies have demonstrated that cell-scaffold composites permit tissue ingrowth and thus readily undergo vascularisation. The novel molecular-interaction mechanism of self-assembly of these scaffolds differentiates this material from other injectable systems. The formation of porous scaffolds within a cavity or a soft-tissue could be a pre-requisite for tissue remodelling using new cell sources that are dependent on vascularisation and tissue ingrowth. The basic component of the scaffold is a biodegradable microparticle that presents a protein resistant surface with biotinylated moieties. Therefore, standard controlled release technologies and biotin-avidin mediated surface engineering can be combined with the self-assembly to form biomimetic scaffolds that stimulate integrin-mediated cell adhesion and then release growth factors.

Conventional methods of combining mammalian cells and synthetic polymers for tissue engineering applications are frequently problematic. This is due to the incompatibility between the sensitive cell component and the harsh polymer processing environments required to form the desired porous scaffold e. g. high temperatures and organic solvents. This results in the necessity for an often inefficient and time consuming two step scaffold seeding process, whereby mammalian cells are added to a pre-fabricated polymer scaffold. High pressure or supercritical CO2 (scCO2) processing is a method of fabricating porous polymer scaffolds at ambient temperatures and without using organic solvents. When pressurised, CO2 becomes highly soluble in a variety of amorphous polymers such as poly(DL-lactic acid) (PDLLA) to produce a high viscosity liquid. Subsequent decompression causes the formation of gas bubbles that become permanent as the polymer vitrifies. Based upon technology at the University of Nottingham, we hypothesised that mammalian cells could be incorporated into poly(DL-lactic acid) (PDLLA) scaffolds using a single step scCO2 process. This would not only make the process more rapid, but it would remove the inefficient scaffold seeding step required in most cell based tissue engineering strategies. Mammalian cells were subject to a range of high pressure CO2 and N2 processing conditions and assessed for cell survival. It was discovered that primary hepatocytes, meniscal fibrochondrocytes, myoblastic C2C12s and 3T3 fibroblasts could survive after exposure to both high pressure gases on a time and pressure dependent basis. Cells exposed to scCO2 for one minute were then assessed for both gene and enzyme function.Using a microarray, it was found that only eight genes (out of 9000) in murine C2C12 cells were significantly down-regulated when compared to untreated cells. Continued cell function was confirmed by measuring BMP-2 induced alkaline phosphatase activity as a measure of osteogenic differentiation in myoblastic C2C12 cells. Alkaline phosphatise activity was indistinguishable between untreated cells and cells exposed to scCO2 for one minute. Additional enzyme and receptor function was confirmed by measuring cytochrome P450 activity in primary hepatocytes after one minute of scCO2 processing. In the second half of the study, these short processing times were found to be sufficient to plasticise and foam porous PDLLA scaffolds. Therefore, cells were incorporated into the biodegradable PDLLA foams by pre-mixing the cell suspension with the polymer powder and exposing to scCO2. Subsequent decompression caused the polymer to foam with the cells trapped within the porous structure. Despite the presence of the plasticised PDLLA, cell survival was confirmed by both an Alamar B1ueTM assay and LIVE/DEADTM staining. Osteogenic differentiation on the scaffolds was confirmed by a stain and assay for BMP-2 induced alkaline phosphatase activity. Finally, a second generation processing piece of processing apparatus was designed that permitted mammalian cells to be passed into a pressurised vessel containing preplasticised PDLLA using a novel high-pressure CO2 injection system. This was made possible by constant optimisation of the high pressure apparatus and the introduction of a cell delivery valve. When injected at high pressures cell survival was found to be reduced when compared with previous experiments although this was likely to be due to the additional mechanical trauma caused by the injection process. Despite this, the live cell population was shown to retain its osteogenic differentiation capacity when induced with BMP-2. With further optimisation of the delivery method, cells may survive this process in sufficient numbers to suggest that it could be used as a method of seeding tissue engineering scaffolds in the future. This development could remove the limitations place on polymer processing time by the finite survival period of the cells, permitting tuning of the scaffold structure to suit the application. In summary, this study has demonstrated that mammalian cells can be incorporated into biodegradable PDLLA scaffolds using a rapid, one-step scCO2 process without the use of toxic solvents or elevated temperatures. Furthermore, the development of the high pressure injection system could allow cells to be incorporated during the fabrication step, removing the restrictions on polymer processing. This technique could be used for the rapid production of tissue cell loaded engineering scaffolds and other associated biotechnological applications where cells and synthetic polymers are combined, such as cell therapy and recombinant protein production.

Damage to cartilage, caused either by disease or injury, affects a large number of people worldwide, severely reducing the patient's quality of life and generating a huge burden on healthcare systems. The limited success of treatment options such as tissue grafts has been the driving force behind much research into tissue engineering strategies for cartilage repair. One of the challenges associated with tissue engineering cartilage is that of generating constructs of clinically relevant sizes since the formation of a crust of tissue at the scaffold periphery restricts the supply of nutrients to the growing tissue. The hypothesis of this thesis was that a tissue engineering system incorporating scaffolds containing both random and anisotropic porosity and a novel flow perfusion bioreactor system would facilitate in vitro tissue formation by enhancing the supply of nutrients to the growing construct. This hypothesis was examined using cartilage as a model tissue. It was shown that scaffolds combining both random and anisotropic porosity (sparse knit scaffolds) had improved flow properties compared to scaffolds containing random porosity alone (needled felt scaffolds). Following studies to characterise the scaffolds and to determine the appropriate conditions for seeding cells into the scaffolds, cartilage formation within the different scaffolds was assessed over a four week culture period. It was found that the flow perfusion system was not as favourable for in vitro cartilage formation as either the commercially available Rotary Cell Culture System (RCCS) or static culture. One of the sparse knit scaffolds (sparse knit 4) and the needled felt were further compared for cartilage formation over an eight week culture period, using static and RCCS culture. With respect to collagen and glycosaminoglycan (GAG) production, cartilage constructs generated from the two scaffold systems were similar. Following static culture it was found that more viable cells were present at the centre of sparse knit 4 scaffolds than needled felt scaffolds. It was therefore concluded that scaffolds combining random and anisotropic porosity were advantageous for culturing tissues in environments where nutrient supply was reliant on diffusion alone.

Embryoid body formation is a commonly used procedure in embryonic stem cell differentiation as it recapitulates the early stages of embryogenesis and in doing so induces the formation of the three germ layers. Despite being a commonly used step in the differentiation of embryonic stem cells there is still a raft of inconsistencies in this process. As a result, heterogeneity exists in the cells produced in terms of their number and differentiation status; an issue which must be overcome in order to realise the full potential of embryonic stem cells for regenerative medicine. The work produced here is focussed on the issue of size heterogeneity during embryoid body formation. First of all, a closer look at the inherent size-dependent characteristics of embryoid bodies was explored over a 120-hour formation period. This showed that the mean diameter and span of embryoid bodies continued to increase throughout the duration of the 120 hours and provided insight into the population dynamics over this formation period. A non-scalable mesh separation technique was produced to further explore the inherent characteristics of embryoid bodies. This was shown to be successful at collecting size fractions of < 100µm, 100-200µm and > 200µm. Immunohistochemistry was used to show size-dependent differences in embryoid body differentiation by showing differential surface expression of Gata-4, Brachyury and Nestin in different sized populations. Meanwhile cell counts and a growth rate assay were performed which showed further size-dependent differences in the ability of each fraction to produce large numbers of cells. This showed that EBs within the 100-200µm fraction contained the largest numbers of cells and were the most proliferative fraction. However, they were also the only fraction to not express proteins from all three germ layers to any level. Due to the clear size-dependent differences in these size fractions perfusion technique was developed to separate embryoid bodies according to their size in a glass column. This depended on the relationship between particle diameter and terminal falling velocity. Key experimental factors that impacted on the efficacy of this technique were shown to be settled bed height, flow rate, sampling height and flow rate. As a result, two artificial neural networks were developed using a model system of Spehadex beads to show how varying these factors impacted the mean diameter and span of collected particles at a constant settled bed height. These experiments showed that this technique was limited to collecting particles from the lower reaches of the stock for both Sephadex beads and embryoid bodies. This was because the most successful factor for increasing mean diameter, flow rate, also induced the greatest increase in span. Therefore, collecting larger sized fractions reduced their discrete nature to such an extent that they were no longer distinct from one another. This suggested that this technique had potential, but was limited by the flow characteristics caused by increasing flow rate. As a result, the separation technique was adapted to overcome this by inverting the column and removing the impact of flow rate; instead allowing gravity alone to separate the particles using a top-loading method. This method was shown to be substantially more successful at collecting larger particles in discrete fractions, however, there were issues with collecting the smaller fractions during a 15-minute separation. Adaptation of this technique allowed all three size fractions to be collected effectively. Finally, a growth rate assay was performed to determine if the embryoid bodies could survive the conditions encountered in this technique and continue to grow post-separation. The cells collected from the column were able to continue growing when dissociated and grown in two-dimensional culture. However, their growth rate was lower than that in the unseparated control and it is not known whether this was because the technique is destructive. This research has therefore identified a cheap, effective, tuneable, novel size-separation technique for embryoid bodies that can collect multiple fractions in a single run with high throughput.