Abstract
Human vocal folds possess outstanding abilities to endure large, reversible deformations and to vibrate up to more than thousand cycles per second. This unique performance mainly results from their complex specific 3D and multiscale structure, which is very difficult to investigate experimentally and still presents challenges using either confocal microscopy, MRI or X-ray microtomography in absorption mode. To circumvent these difficulties, we used high-resolution synchrotron X-ray microtomography with phase retrieval and report the first ex vivo 3D images of human vocal-fold tissues at multiple scales. Various relevant descriptors of structure were extracted from the images: geometry of vocal folds at rest or in a stretched phonatory-like position, shape and size of their layered fibrous architectures, orientation, shape and size of the muscle fibres as well as the set of collagen and elastin fibre bundles constituting these layers. The developed methodology opens a promising insight into voice biomechanics, which will allow further assessment of the micromechanics of the vocal folds and their vibratory properties. This will then provide valuable guidelines for the design of new mimetic biomaterials for the next generation of artificial larynges.
Highlights
Human vocal folds exhibit remarkable vibro-mechanical properties, allowing them to generate an outstanding range of sounds
A loose connective tissue called lamina propria (LP, thickness ≈1–2.5 mm), made of cells and extracellular matrix (ECM) with amorphous ground substances and fibrous networks
Since the reference histological findings and 3D sketches on the vocal-fold multilayered arrangement provided by Hirano et al in the 1970-80s6,41,42, very few experimental quantitative data have been collected to characterise the complex architecture of human vocal-folds, in particular at the micrometer scale
Summary
After a 3-day immersion in an aqueous solution with 30% of ethanol (Supplementary Fig. S2(b)), the different vocal-fold sublayers can be identified, i.e., the vocalis with well-defined cross sections of muscle fibres, the lamina propria with a similar but finer texture, and the epithelium with some saturated brightness due to sharp phase contrast at air-tissue interface. (a) 3D orientation map of the collagen and elastin fibrous networks in a LP-layer 0.018 mm[3] subvolume derived from microtomographic measurements acquired at high spatial resolution (voxel size of 0.653 μm3); corresponding. Due to the limited contrast obtained in this zone (see Fig. 4), no threshold-based segmentation was successfully achieved to provide a statistical quantification of their dimensions and shape
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