Fig 1.
Schematic of larynx and vocal folds for growth measurements.
(a) Laryngeal transverse diameter. (b) Laryngeal length. (c) Laryngeal outer dorsoventral diameter (d) Vocal fold thickness. (e) Vocal fold internal length.
Fig 2.
Schematic of AFM sampling for biomechanical stiffness.
Probe is placed on lamina propria and repeated indentation measurements are taken.
Fig 3.
Growth measurements for larynx and vocal folds.
(a) Graph of larynx measurements in dorsoventral diameter, larynx length, and transverse diameter. Quadratic growth patterns were found for all measurements. Comparisons of larynx length time points report statistically significant changes between all sequential time points except E13.5-E15.5, E16.5-E18.5, and E18.5-P0. Outer dorsoventral diameter was significant between all sequential time points except E13.5-E15.5 and E18.5-P0. Transverse diameter was significant between all sequential time points except E13.5-E15.5 and E18.5-P0. (b) Graph of measurements of the vocal fold in internal length and thickness, shown by time point. Internal VF length increases linearly while VF thickness increases quadratically. Comparisons of VF measurement time points reported significant changes between all internal length time points except E13.5-E15.5 and E18.5-P0. Thickness was significant between all sequential time points except E15.5-E16.5 and E18.5-P0.
Table 1.
Inter- and intra-reliability for larynx and VF measurements.
Fig 4.
Proliferation measurements in transverse and coronal VF planes.
(a) An EdU assay was performed to determine cellular proliferation in the laryngeal region. Coronal and transverse sections were taken at each time point. Cartilage cells were excluded from proliferation counts. Proliferation rates were significantly higher in early stages than at late stages. VF = vocal folds, bright green cells are those that are proliferating. (b) Graph of proliferation rates in the coronal and transverse VF, shown by time point. High decreases in proliferation are shown in early embryonic stages, and proliferation plateaus and stops by adult stages. Sequential comparison of coronal plane proliferation time points reported significant changes between E13.5-E15.5. Sequential comparison in the transverse plane reported significant differences between E13.5-E15.5 and E15.5-E16.5.
Fig 5.
Graph of vocal fold stiffness, shown by time point.
Stiffness was measured using atomic force microscopy. Statistical analysis found stiffness was found to increase quadratically over time. Large increases in variance evident with increase in time.