Multispectral three-dimensional optical coherence tomography

A spectral-domain OCT system operating at 1300 nm wavelength region, capable of acquiring 47,000 A-lines/s, was designed and developed. Its axial and transverse resolutions were 6 micro and 15 &micro respectively. OCT images of human skin were obtained in vivo using three OCT systems, in order to find the optimal wavelength region for dermal imaging. 800 nm OCT system provided better image contrast over other two wavelength regions. Meanwhile, 1300 nm wavelength region was needed to obtain information from deeper dermal layers. To determine the effect of melanin pigmentation on OCT, images were taken from subjects with different ethnic origins. Interestingly, melanin pigmentation was found to have little effect on penetration depth in OCT. In vitro tumour samples, comprising samples with different degrees of dysplasia, were imaged at 800 nm, 1060 nm and 1300 nm wavelength regions to find the capability of OCT to diagnose microstructural changes occurring during tumour progression. 800 nm OCT system was capable to detect the malignant changes with higher contrast than other wavelength regions. However, higher wavelength regions were required to penetrate deeper in densely scattering tumour samples at advanced stages. OCT system operating at 1060 nm was combined with a photoacoustic imaging (PAT) system to obtain complementary information from biological tissues. This multimodal OCT/PAT system demonstrated its potential to deliver microstructural information based on optical scattering and vascular information based on optical absorption in living mice and human skin. The results indicate OCT as a promising imaging modality that can have profound applications in several areas of clinical diagnostic imaging

The Clinical Application of Optical Coherence Tomography for Head and Neck Premalignant/Malignant Lesions

Abstract The principle of Optical Coherence Tomography (OCT) is based on the property of light coherence. OCT generates cross-sectional images of two-dimensional objects to obtain in-vitro and in-vivo images of tissues. Non–commercially available OCT systems, which have a higher resolution and scanning rate, have been previously reported. However, some clinical research has already been conducted using the first commercially available OCT device (Niris system) to image the larynx; but applications on oral and skin tissue have not been tested yet. This thesis aims to explore, compare and validate three specific types of commercially available OCT equipment for imaging head and neck tissue. An animal cancer model has been used to verify the feasibility of one system (Niris) to differentiate normal from malignant oral tissue, using in-vivo tissue samples. Since images of oral tissue samples didn’t show much structure using the Niris system, a different machine (Michelson Diagnostic bench based) with different specifications and resolution was employed. Great emphasis has been put on validating OCT structurally and histomorphometrically in comparison to the gold standard of pathology. This was tested and validated with ex-vivo oral and skin tissues using the lab based version of the machine. Use of an upgraded system (Michelson vivo sight with probe) has been tested on abnormal oral and skin biopsy tissue but with different timing for the scan (instant ex- vivo). One original study evaluated and classified tongue papilla atrophy from patients having their suspicious tongue lesions biopsied. In conclusion, this thesis concludes that the new version of this commercially approved OCT system can be applied to the diagnosis of superficial premalignant and malignant oral and skin lesions in-vitro. Furthermore, OCT holds the promise of complementing surgery to eradicate tumors and monitor the consequences

Novel insights into hair structure and the effects of chemical stressors on hair and skin using label-free advanced light microscopy

There is a need for a better method to image hair as the current methods involve embedding the hair in resin, which may produce artefacts, or using dyes which are limited in their depth of penetration into the hair. The research performed in this thesis endeavours to characterise the cellular structure of human hair with label-free imaging using autofluorescence and fluorescence lifetime imaging. Wavelengths were shown to selectively excite the hair cuticle, cortex and medulla, and subcellular compartments. Development of an optical transverse imaging method enabled discoveries including different fluorescence lifetimes across the cuticle cell layers and suggests the cuticle layers possess differing chemical environments. A new method was developed to distinguish between eumelanin and pheomelanin using 405nm and 633nm wavelengths. The newly developed methods were additionally used in the characterisation of an unidentified hair and skin disorder, which found poorly differentiated cuticle cells and showed differences in the fluorescence lifetimes of the hair compared to control hairs. The hair care industry needs more efficacious chemical depilatories and information into their action. This was elucidated using the developed methods and a new dynamic imaging method. Potassium thioglycolate was shown to cause drastic expansion of the hair which was amplified by the addition of guanidine carbonate, creating fissures through the cuticle and into the cortex. Other experimental depilatory formulations were tested and were found to have varying effects upon the structure of the hair. New chemical depilatories require development because existing depilatories can cause irritation in the skin. Potassium thioglycolate and guanidine carbonate were tested on HaCaT cells, isolated cornified envelopes, and HEKn cells in a 3D epidermal model. An investigation into the differentiation, proliferation and acute stress response of the cells showed that the treatments had no significant effect on these markers. However, the chemicals negatively affected HaCaT cell viability and damaged the cornified envelopes. Despite this, the viability and structural integrity of the living cells of the epidermal model were maintained through the protection provided by the stratum corneum

A novel biodegradable poly(ε-caprolactone urea)urethane incorporating polyhedral oligomeric silsesquioxane nanocomposite and applications for skin tissue engineering

Skin protects our bodies for a lifetime and extensive loss of this barrier renders the individual susceptible to infections and death. Clinically available treatment options, however, are limited in establishing both functional and cosmetic satisfaction. The work described in this thesis is therefore concerned with the development and characterization of a novel biodegradable nanocomposite system displaying suitable properties as skin tissue engineering scaffolds. A novel family of segmented polyurethanes (PU) with increasing hard segment content based on a poly(ε-caprolactone urea)urethane backbone incorporating POSS nanoparticles was synthesized and analysed in terms of material characteristics and biocompatibility. Incorporation of POSS nanoparticles into the PU backbone yielded mostly amorphous materials as corroborated by distinct glass transitions visible on differential scanning calorimetry spectra. With incrementally increasing hard segment content, ultimate tensile strength increased from ~10 to 21 MPa accompanied by increased values for elastic moduli from 0.03 to 0.06 MPa. Number average molecular weights (Mn) decreased with increasing hard segment content due to a corresponding decrease in the proportion of PCL. Sterilization studies raised fundamental concerns regarding the suitability of conventionally available techniques since hydrolytically and temperature labile polymers are susceptible to degradation. The results obtained suggest 70 % ethanol to be a suitable laboratory-based disinfectant which was further demonstrated to have favourable effects on skin cell compatibility. Degradation studies revealed hard segment-dependent modulation of the degradation rate and the materials’ viscoelasticity. In vivo implantation studies of porous scaffolds demonstrated firm integration with the subcutaneous tissue and extensive vascularization. The results obtained in this work highlight (i) the ability to control scaffold degradation rates and mechanical properties and (ii) cellular as well as in vivo biocompatibility, all of which fundamental in the development of a versatile skin tissue engineering scaffold

Aryl hydrocarbon receptor activation in primary human keratinocytes and epidermal equivalents

The Aryl hydrocarbon Receptor (AhR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulting in the human specific toxicity, chloracne. To test whether the chloracnegenic potential of AhR-agonists depends upon binding affinity for the AhR, residency and/or down-regulation of the AhR, we investigated the effects of different AhR agonists in primary human keratinocytes and epidermal equivalents. The AhR agonists used were high-affinity, high-residency and high-potency TCDD, and two agonists not known to induce chloracne; low-affinity, low-residency and low-potency β-naphthoflavone (β-NF) and the low-affinity, low-residency and high-potency physiological agonist 2-(1‟H-indole-3‟-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). -NF, a partial agonist was used to test AhR dependency. The effects of these agonists on AhR activation, terminal differentiation, autophagy and expression of cathepsin D (CTSD) in primary human keratinocytes and epidermal equivalents were determined. All three agonists induced AhR activation by XRE-luciferase assay, which was inhibited by α-NF, demonstrating AhR dependence of the ligands. AhR degradation was induced by all ligands and CYP1A1 was induced strongly by TCDD but weakly by β-NF and ITE. CYP1A1 and XRE-luciferase induction correlated with ligand binding affinity; ranking levels of binding affinity as TCDD>β-NF>ITE. TCDD treatment induced a chloracne-like phenotype in epidermal equivalents, with a decrease in viable cell layer thickness and compacted stratum corneum. This was not induced by β-NF or ITE. To investigate the differential effects of AhR-ligands on epidermal equivalent phenotype, we studied differentiation markers filaggrin, involucrin and TGM-1. TGM-1 expression was induced specifically by TCDD while aberrant expression of involucrin and filaggrin were induced by TCDD, β-NF and ITE. AhR activation was not associated with increased apoptosis. Caspase-3 independent cell death has been implicated as a mechanism of decreased thickness of the viable cell layer, so we studied the effects of AhR-agonists on autophagy. Autophagy in keratinocytes and epidermal equivalents was characterised by induction of LC3 II, p62 degradation and transmission electron microscopy. TCDD robustly induced active autophagy, while ITE induced lower levels and β-NF blocked autophagy. TCDD- and ITE-induced autophagy in epidermal equivalents appeared to result in decreased numbers of lamellar bodies, which may account at least in part for the compacted stratum corneum phenotype shown by the TCDD-induced phenotype in epidermal equivalents and chloracne. As CTSD has been implicated in keratinocyte differentiation and an XRE domain has been identified upstream of CTSD, we studied the effects of ligand-dependent AhR activation on lysosomal aspartic protease CTSD expression. CTSD was increased by AhR activity in epidermal equivalents. Induction of CYP1A1 did not appear to be a specific biomarker of chloracnegenic potential of AhR agonists. The data presented have shown differential effects by TCDD, β-NF and ITE on autophagy that we hypothesise contributes to the chloracne phenotype. In this thesis, potential biomarkers specific to chloracne were identified in keratinocytes, TGM-1, CTSD, autophagy and decreased lamellar bodies, although further validation is required.EThOS - Electronic Theses Online ServiceBBSRCAstraZenecaGBUnited Kingdo