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Raynaud’s phenomenon (RP) describes excessive vasoconstriction of the digital microvasculature in response to cold exposure and emotional stress. RP is typically the earliest clinical manifestation of systemic sclerosis (SSc); a complex multisystem disease of unknown aetiology characterized by vasculopathy, inflammation and fibrosis. Vasculopathy is considered an essential pre-requisite to tissue remodeling, characterized by excessive collagen synthesis and tissue fibrosis, which occurs in the skin and other organs of patients with SSc. There is mounting evidence associating platelets with important biological functions beyond primary haemostasis. Upon activation, platelets release a large array of mediators implicated in vasoconstriction, inflammation and fibrosis, which has led me to consider the contribution of platelets to the pathogenesis of both RP and SSc. The principal aim of this thesis is to explore the impact of targeted anti-thrombotic therapy on digital microvascular function, platelet activation, oxidative stress and eicosanoid biosynthesis in RP and SSc. It is not possible to examine the impact of anti-platelet therapy on microvascular function, without first identifying sensitive methods for assessing digital microvascular function in humans. I shall report the findings of work examining established (infrared thermography) and novel (laser speckle contrast imaging) methods for the objective assessment of digital microvascular function in RP and SSc. I shall critically appraise the application of these methods alongside subjective patient self-report assessment of digital vascular function. I shall examine associations between digital vascular function, platelet activation, oxidative stress and eicosanoid biosynthesis between primary RP and SSc. I shall also report the findings of an investigator-led early phase clinical trial of targeted anti-thrombotic medication in RP and SSc. The major findings of this study were highlighting the strengths and limitations of established and novel methods for objective microvascular assessment in RP and SSc, and the poor agreement that exists between objective and subjective methods for assessing RP severity. I have identified similarities and differences in platelet activation and eicosanoid biosynthesis in primary RP and SSc. I shall present evidence of apparent efficacy of asasantin retard therapy on subjective, but not objective, assessments of digital microvascular function; the relevance of which shall be discussed in detail.

The aim of this work was to improve the utility of hollow fibre bioreactors (HFB) for the growth of clinically applicable cell populations by improving initial rates of cell attachment using a novel dynamic seeding methods, and to investigate their use as a novel cell encapsulation device for the delivery of cell therapies to infarcted heart tissue. Cardiac cell therapies starting are traditionally administered by direct injection or intravenous infusion to a target area in the heart. Mesenchymal stem cells (MSCs) have shown promise as a cell therapy and can be isolated from a patient’s adipose tissue surrounding the heart, allowing 812,800 ± 318,400 cells to be harvested; however this population requires expansion to a minimum of 50 million cells to be an effective traditionally administered dose in humans. Hollow fibre bioreactors have shown to offer the highest rates of MSC growth compared to other bioreactor designs, owing to their large surface area to volume ratio and their ability to provide the cells with a low shear environment that can emulate in vivo conditions. However, a concern with using HFBs is the relatively low rates of initial cell attachment, with authors observing rates around 20%. At this rate of attachment and with an initial population of 812,800 ± 318,400 cells, it would take 12.6 ± 5.2 days to grow 50 million MSCs in a HFB. For use as a medical intervention the MSCs must be grown as soon as possible, and to ensure this the efficiency of cell seeding on the external hollow fibre surface must be as high as possible. In an attempt to improve rates of initial cell attachment Chapter 4 illustrated how dynamic seeding can allow additional opportunities for a given cell to make contact with hollow fibres contained in a bioreactor. MG63 human osteosarcoma cells were used as an alternative cell type due to their lower cost and increased resilience over a greater number of passages. Static horizontal seeding of MG63 cells on single fibre bioreactors provided 18% ± 16% cell attachment confirming values found in literature, however dynamic attachment on horizontally orientated multi fibre bioreactors rotating at 6 RPM offered improved attachment of 39% ± 12%, double the rate of static attachment. At this improved rate of cell attachment, it would now take 11.2 ± 4.7 days to grow 50 million cells, 1.4 days quicker than before. This novel approach to HFB seeding will lead to quicker administration of medical treatments for patients. While traditional delivery methods have shown to improve heart function, the active dose retained at the wound site is around 2%, much lower than that actually administered. Cells can be ejected from the insertion site due to heart contractions, and doses contained in the bloodstream become highly diluted. Engineered alternatives include cellular sheets, cell-loaded sutures, and cell encapsulation within hydrogels. However, each of these have their own inherent limitations, with cellular sheets demonstrating poor rates of migration into infarct site, cells on suture surfaces shearing away when sown into a wound site, and cells in hydrogels suffering from poor initial rates of engraftment. Hollow fibres made from poly(lactic-co-glycolic acid) (PLGA) were further investigated for use as an engineered cell delivery vector, with cells seeded within the hollow fibre lumen, allowing cells to be protected during implantation, biodegrade, and provide a concentrated cell dose at a wound site. Hollow fibre scaffolds have been used as a delivery device for encapsulation of pharmaceuticals, but never for encapsulation of cells in this manner. Chapter 5 investigates the development of a hollow fibre with internal and external surface porosity, which allowed passive permeation of liquid through its structure from one side to the other. Manufacturing a hollow fibre using NaCl porogen at 60% the weight of PLGA resulted in pores 4 – 6 μm in diameter, which provided the desired liquid permeation characteristics and a flux of 6.6 m3m-2h-1bar-1 (± 77%). Chapter 6 sought to encapsulate a population of MG63 cells within the lumen of the hollow fibre, fed by permeation of media flowing in the extracapillary space (ECS), and investigate the viability of cells over a 6 day time course. Studies revealed that the cells became non-viable after 24 hours as a result of poor rates of dissolved oxygen permeation. Subsequent modelling of the system showed that an increased ECS flow rate of a 1.49 mL/min should provide sufficient dissolved oxygen to sustain a population of 100,000 cells in this system. This work demonstrates a vital first step in exploring a potential evolution in cell delivery, with further investigation required to fully explore the viability of hollow fibre devices for cell encapsulation.

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