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

Multiplexed High-Resolution Imaging Approach to Decipher the Cellular Heterogeneity of the Kidney and its Alteration in Kidney Disease and Nephrolithiasis

Indiana University-Purdue University Indianapolis (IUPUI)Kidney disease and nephrolithiasis both present a major burden on the health care system in the US and worldwide. The cellular and molecular events governing the pathogenesis of these diseases are not fully understood. We propose that defining the cellular heterogeneity and niches in human and mouse kidney tissue specimens from controls and various models of renal disease could provide unique insights into the molecular pathogenesis. For that purpose, a multiplexed fluorescence imaging approach using co-detection by Indexing (CODEX) was used, using a panel of 33 and 38 markers for mouse and human kidney tissues, respectively. A customized computational analytical pipeline was developed and applied to the imaging data using unsupervised and/or semi-supervised machine learning and statistical approaches. The goal was to identify various cell populations present within the tissues, as well as identify unique cellular niches that may be altered with disease and/or injury. In mice, we examined disease models of acute kidney injury (AKI) and in human tissues we analyzed specimens from patients with AKI, IgA nephropathy, chronic kidney disease, systemic lupus erythematosus, and nephrolithiasis. In both mice and humans, the disease and reference samples show similar broad cell populations for the main segments of the nephron, endothelium, as well as similar groups of immune cells, such as resident macrophages and neutrophils. When comparing between health and disease, however, a change in the distribution of few sub-populations occurred. For example, in human kidney tissues, the abundance and distribution of a subpopulation of proximal tubules positive for THY1 (a marker of differentiation and repair), was markedly reduced with disease. Changes observed in mouse tissues included shifts in the immune cell population types and niches with disease. We propose that our analytical workflow and the observed changes in situ will play an important role in deciphering the pathogenesis of kidney disease