Events

Date: 3 December 2024   Time: 12:00 - 12:30

Location: SEMS Seminar Room, 3rd floor, Engineering Building

Title:

TomoSAXS: combining small-angle scatter and X-ray tomography for hierarchical biomechanics of collagenous tissues

Talk Summary of Dr Elis Newham:

Collagenous tissues, key constituents in the overwhelming majority of vertebrate biomechanical systems, consist of a range of structures and assemblies that operate hierarchically over spatio-structural scales. This creates a complex interplay between nanoscale and microscale biomechanics that is vital for understanding the function of organs such as the intervertebral disc (IVD), and the effects of ageing and disease. We have developed "TomoSAXS", a method for characterising these relationships through bimodal synchrotron X-ray probing combining tomographic small angle X-ray scattering (SAXS) and microtomographic X-ray imaging (μCT). Unlike previous techniques for volumetric SAXS analysis, TomoSAXS uses μCT to estimate the angular orientation of nanoscale collagen structures, permitting relatively rapid SAXS tomographies over a single axis and allowing performance of biomechanical experiments during scanning. These orientations are used to simulate the SAXS tomography and estimate instances of independent SAXS scatter for individual collagen fibres using three-dimensional (3D) diffraction modelling, validated for accuracy using simulations of digital phantoms. Sampling and modelling of these instances in real data permits characterisation of structural and mechanical properties for fibres over volumetric samples across whole tissues beyond the SAXS sampling limit, here the IVD. Analysis of intact IVD under physiological compressive loading allows further use of μCT for digital volume correlation (DVC), which is in-turn used to calibrate the identity of fibres in TomoSAXS reconstructions before and after loading. This allows estimation of per-fibre nanoscale strain, and direct correlation of mechanics between the nano-to-micro scales across whole organs.

https://scholar.google.com/citations?hl=en&user=JjLPWoEAAAAJ&view_op=list_works&sortby=pubdate

Title:

Development of a functional 3D eccrine sweat gland model

Talk Summary of Holly Bachas Brook:

Human skin is populated by a uniquely high density of eccrine sweat glands (ESGs), which are primarily responsible for thermoregulation. Eccrine sweat also contains moisturizing factors and antimicrobial peptides, which contribute to maintaining a healthy skin barrier. To date, the absence of functional, non-animal models to study the human ESG is a significant challenge to researchers. This project aimed to produce a microfluidic ESG-on-chip model that mimics ESG functionality. Microfluidic polydimethylsiloxane (PDMS) chips were fabricated via soft lithography, comprising three parallel channels separated by microposts; bonded to a glass coverslip via oxygen plasma. The central hydrogel-filled channel mimicked dermal ECM, with two parallel channels on either side for the ESG tubule and media perfusion, respectively. EC23 is a novel human ESG cell-line, which formed a confluent tubule within 24 hours of microfluidic culture. Retention of FITC-dextran within the cell-lined channel confirmed the establishment of an epithelial barrier. Functional ESG proteins, including muscarinic receptor 3 (M3), aquaporin 5 (AQP5), Na-K-Cl cotransporter 1 (NKCC1), anoctamin-1 (ANO1), and stromal interaction molecule 1 (STIM1), were upregulated within the ESG-on-chip. Analysis of calcium flux in the ESG-on-chip was also investigated using intracellular calcium indicator Fluo8. Stimulating with the M3 agonist carbachol increased intracellular calcium in a dose-dependent manner. Additionally, an intracellular pH (pHic) assay demonstrated NKCC1-mediated ion transport by examining pHic recovery after treatment with ammonium chloride. The rate of pHic recovery (ΔpH/second) increased following pre-treatment with carbachol, which stimulates NKCC1 activity via an increase in intracellular calcium. Conversely this response was blocked by the specific NKCC1 inhibitor bumetanide. This ESG-on-chip model replicates key structural, molecular, and functional aspects of the human ESG. The ESG model and quantitative assays established here represent new experimental tools for assessing human ESG functionality in response to drugs, chemicals, or environmental changes in vitro.

https://www.cpm.qmul.ac.uk/people/hbachasbrook/