Raman spectral imaging in tissue engineering & regenerative medicine applications

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.Open Acces

Quantitative volumetric Raman imaging of three dimensional cell cultures

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy

Fibres and cellular structures preserved in 75-million-year-old dinosaur specimens

Exceptionally preserved organic remains are known throughout the vertebrate fossil record, and recently, evidence has emerged that such soft tissue might contain original components. We examined samples from eight Cretaceous dinosaur bones using nano-analytical techniques; the bones are not exceptionally preserved and show no external indication of soft tissue. In one sample, we observe structures consistent with endogenous collagen fibre remains displaying ∼67 nm banding, indicating the possible preservation of the original quaternary structure. Using ToF-SIMS, we identify amino-acid fragments typical of collagen fibrils. Furthermore, we observe structures consistent with putative erythrocyte remains that exhibit mass spectra similar to emu whole blood. Using advanced material characterization approaches, we find that these putative biological structures can be well preserved over geological timescales, and their preservation is more common than previously thought. The preservation of protein over geological timescales offers the opportunity to investigate relationships, physiology and behaviour of long extinct animals

Quantitative volumetric Raman imaging of three dimensional cell cultures

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy

Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate

Cell size and shape affect cellular processes such as cell survival, growth and differentiation1,2,3,4, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications