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Title: Raman spectral imaging in tissue engineering & regenerative medicine applications
Author: Kallepitis, Charalambos
ISNI:       0000 0004 7963 6879
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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The label-free nature of Raman spectroscopy makes it a valuable tool for cellular and tissue characterisation. Its ability to probe molecular vibrations within biological structures without affecting their biochemistry offers an advantage over conventional histological and biochemical assays. Providing a pure investigation of unperturbed biological processes, without the need for introduction of exogenous molecules for labelling, makes the information Raman spectroscopy offers very valuable in deciphering complex biological functions and mechanisms. Raman spectral signatures are unique "fingerprints" of each biomolecule probed and can be used for cellular phenotype characterisation, tissue composition, disease development in a cellular or tissue level and much more. This thesis focuses on the use of Raman spectral imaging in novel biological applications displaying its flexibility across the fields of tissue engineering and regenerative medicine. Bone regeneration was the first biological process investigated, where Raman spectral imaging was used to characterise bioactive glass-assisted bone repair using standard and novel glass compositions. Newly-formed bone quality was assessed using multivariate analysis, showing similar quality between glass compositions and existing bone. Morphological analysis after in vivo implantation of bioactive glass particles showed distinct spectral zones confirming results from existing in vitro models. The second application, focused on the development of a novel Raman-based gene delivery tracking methodology. Viral particles, containing modified viral-nucleotides with alkyne bonds were produced were successfully detected using Raman spectral imaging in cells after infection. The implications of this technology offer a new cell screening methodology for gene therapy. Finally, the potential of Raman spectral imaging as a complementary technique for 3D cell culture systems was explored. A computational framework was developed which allows for the visualisation and quantification of subcellular structures. The accurate 3D reconstruction of whole cells of known architecture from a volumetric hyperspectral Raman dataset was reported here for the first time. Moreover, using spectral unmixing algorithms to quantify subcellular components, revealed an unprecedented molecular specificity. This allowed imaging of cells within hydrogel-based 3D cell culture systems. The synergy of Raman spectral imaging, multivariate and image analysis to answer complex biological questions offers objective biomolecular characterisation, quantification and visualisation of molecular architecture. This work demonstrates the potential of Raman spectroscopy as a valuable complementary tool in tissue engineering and regenerative medicine applications.
Supervisor: Stevens, Molly ; Payne, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral