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The production of human material for skin replacement

Underwood, Sarah Anne; (1999) The production of human material for skin replacement. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

This thesis describes the research to identify a process for the production of cell guidance scaffolds with potential use as human skin replacements. Fibronectin, a plasma protein with well-documented cell adhesion properties, can be extracted from the cryoprecipitate collected from human plasma donations. Purified solutions of fibronectin have been used to make mats with approximate dimensions 1 × 2 cm and consisting of orientated, protein fibrils with diameter 5–10 µm. These mats have demonstrated excellent in vitro and in vivo cell guidance properties. The aim of this research was to produce scaffolds with the same cell guidance / adhesion properties as the mats combined with a simple manufacturing method, little material wastage, reproducibility and potential for large scale production. Polyethylene glycol fractionation was used to produce fibronectin enriched precipitate from cryoprecipitate, with fibronectin and fibrinogen in a 65 : 35 percentage ratio. The precipitation of fibronectin and fibrinogen with a reduction in pH was examined and it was found that fibronectin / fibrinogen cables could be drawn up from a solution when protein was precipitated with 0.1 M citric acid, final pH 4.0–4.5. These cables, average diameter 200 µm, were found to consist of numerous micron-diameter fibrils giving the cable ultrastructural orientation. The cables were found to be hygroscopic and could be stabilised against solubilisation by severe dehydration. Cables which were twisted as they were drawn upwards from solution had an average ultimate tensile strength of 61 N/mm2 , but were brittle. Although these cables could be made easily and were found to have good cell adhesion properties, drawing upwards from solution is not a technique applicable to large scale manufacture. Development of wet-spun fibres using traditional textile manufacturing techniques required that both the solution viscosity and protein concentration were increased. Urea was used as a solvent to increase protein concentration and sodium alginate was used to increase the viscosity of the solution and alter its flow behaviour. A variety of coagulation baths were tested in order to find the optimal pH and salt concentrations for fibre formation. Fibres spun on a pilot scale wet-spinning rig, diameter 500 µm, were found to have properties which varied with their composition and post spinning treatments. The fibres had a lower tensile strength, 28 N/mm2, than the drawn, twisted cables but had a higher elongation at break, 52.4% compared to 3.5%, making them easier to handle. Wet spinning has been shown to be a suitable and flexible process for the production of fibronectin / fibrinogen fibres. The production of fibres is considered with respect to process design, virus inactivation and GMP considerations. A test of the fibronectin material's ability to support the attachment and alignment of human dermal fibroblasts is also described.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The production of human material for skin replacement
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Applied sciences; Skin replacement
URI: https://discovery.ucl.ac.uk/id/eprint/10099265
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