Strange, Adam Philip; (2019) Quantitative Nanohistology: Collagen Disorders of Connective and Mineralised Tissues. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis explores the behaviour of collagen; both as a biomaterial and as a tissue vulnerable to disease. It aims to develop a novel, scalable, and multimodal system for recognising and characterising collagen abnormalities. Initially, collagen implants were characterised using Scanning Electron Microscopy (SEM), before a more detailed analysis using Atomic Force Microscopy (AFM) and Optical Coherence Tomography (OCT) gave an overview of the mechanical properties and degradation rates. The rates agreed with previously established models, but imaging analysis led to new developments in the manufacturing process. Using an AFM bead functionalised with fibroblasts and characterising ex vivo implants from mice, helped monitor the behaviour and lifespan of the implants. Moving from an engineering biomaterial to ex vivo tissue, mice tendons were obtained. The tendons were induced with a model of the terminal condition ARC syndrome. SEM of ARC syndrome tendons showed disorder throughout the hierarchy, and nanoscale imaging revealed a previously unreported swelling of the D-band in collagen, these findings were highly indicative of disorder. Having confirmed the D-banding swelling, a significant decrease of the Young’s modulus of the tendons was found using AFM nanomechanics. Having confirmed the suitability of AFM on tendons, healthy human skin biopsies were obtained. These were used to form a baseline for healthy skin behaviour at the nanoscale. The addition of histological microscopy using picro sirus red stain allowed for more reproducible AFM measurements. Data analysis of the AFM images was significantly improved through the development of an image quantification protocol. This protocol allowed for rapid comparison of images based on a four-point dominant feature system. Finally, this enhanced imaging and mechanical analysis system, termed Quantitative Nanomechanics (QNM), was applied to three patients with the fibrotic disorder scleroderma and matched healthy donors. Significant changes to the morphology of scleroderma collagen, along with an increase in Young’s modulus was reported.

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