High-frequency viscoelastic shear properties of vocal fold tissues: implications for vocal fold tissue engineering

Abstract

The biomechanical function of the vocal folds (VFs) depends on their viscoelastic properties. Many conditions can lead to VF scarring that compromises voice function and quality. To identify candidate replacement materials, the structure, composition, and mechanical properties of native tissues need to be understood at phonation frequencies. Previously, the authors developed the torsional wave experiment (TWE), a stress-wave-based experiment to determine the linear viscoelastic shear properties of small, soft samples. Here, the viscoelastic properties of porcine and human VFs were measured over a frequency range of 10-200 Hz. The TWE utilizes resonance phenomena to determine viscoelastic properties; therefore, the specimen test frequency is determined by the sample size and material properties. Viscoelastic moduli are reported at resonance frequencies. Structure and composition of the tissues were determined by histology and immunochemistry. Porcine data from the TWE are separated into two groups: a young group, consisting of fetal and newborn pigs, and an adult group, consisting of 6-9-month olds and 2+-year olds. Adult tissues had an average storage modulus of 2309±1394 Pa and a loss tangent of 0.38±0.10 at frequencies of 36-200 Hz. The VFs of young pigs were significantly more compliant, with a storage modulus of 394±142 Pa and a loss tangent of 0.40±0.14 between 14 and 30 Hz. No gender dependence was observed. Histological staining showed that adult porcine tissues had a more organized, layered structure than the fetal tissues, with a thicker epithelium and a more structured lamina propria. Elastin fibers in fetal VF tissues were immature compared to those in adult tissues. Together, these structural changes in the tissues most likely contributed to the change in viscoelastic properties. Adult human VF tissues, recovered postmortem from adult patients with a history of smoking or disease, had an average storage modulus of 756±439 Pa and a loss tangent of 0.42±0.10. Contrary to the results of some other investigators, no significant frequency dependence was observed. This lack of observable frequency dependence may be due to the modest frequency range of the experiments and the wide range of stiffnesses observed within nominally similar sample types.

FIG. 1.

Amplification factor (M) versus frequency for a representative porcine vocal fold sample averaged over three repeats. Symbols are the experimental data points, and the line is the least-squares linear viscoelastic model fit. Error bars are the standard deviation from three repeats on one sample. Inset shows the sample sandwiched between the two hexagonal acrylic plates. Color images available online at www.liebertpub.com/tea

FIG. 2.

Storage modulus and loss tangent for all porcine samples tested as a function of (A) frequency and (B) maximum strain. Data points are an average of all samples from one larynx, and the error bars are one standard deviation of these samples, with only one side shown for clarity. Filled symbols represent the storage modulus (G₁, left axis), whereas the open symbols represent the loss tangent [tan(δ), right axis]. The abbreviations in the figure indicate the age of the tissues: 2+ YO: 2+ years old; 6–9 MO: 6–9 months old; 3–4 MO: 3–4 months old. Color images available online at www.liebertpub.com/tea

FIG. 3.

Storage modulus and loss tangent for all adult porcine specimens tested with the rheometer (A) and the averaged rheometer data presented with the torsional wave experiment (TWE) data (B). Symbols in (A) denote three different pig tissues, and the error bars are one standard deviation of all samples from one larynx. In (B), data at low frequencies (below 20 Hz) are from the commercial rheometer, and data in gray with the point-down triangle (▾) are deemed inaccurate, as per the Discussion section. Error bars on the rheometer data are one standard deviation of all data, whereas error bars for the TWE data are for all samples from one larynx. Filled symbols represent the storage modulus (G₁, left axis), whereas the open symbols represent the loss tangent [tan(δ), right axis]. Color images available online at www.liebertpub.com/tea

FIG. 4.

Cryosectioned tissues samples stained with hematoxylin and eosin (H&E), Alcian blue, and Movat's pentachrome. Images show fetal, 6–9-month-old, and 2+-year-old porcine VFs after the TWE. All cryosections for each age group are from the same specimen. Cell pockets are circled in the Alcian blue image of the fetal tissue. Scale bar for all images: 200 μm. Color images available online at www.liebertpub.com/tea

FIG. 5.

H&E, Alcian blue, and Movat's pentachrome staining of fetal (A), 6–9-month-old (B), and 2+-year-old (C) porcine VF cryosections. (A) The epithelium, the lamina propria, and the vocalis muscle are labeled as 1, 2, and 3, respectively. Circled regions indicate intense glycosaminoglycan staining. (B) The last row shows laryngeal glands further down in the VF. Tissue protrusion at the edge is circled, and laryngeal glands are boxed. (C) Tissue protrusion at the edge is circled. Scale bar: 500 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

Storage modulus and loss tangent for all human samples tested as a function of frequency (A) and the maximum strain (B). Data points are an average of all samples from one larynx, and the error bars are one standard deviation of these samples, with only one side shown for clarity. Filled symbols represent the storage modulus (G₁, left axis), whereas the open symbols represent the loss tangent [tan(δ), right axis]. Color images available online at www.liebertpub.com/tea