Journal of Otology & RhinologyISSN: 2324-8785

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Research Article, J Otol Rhinol Vol: 5 Issue: 3

The Anisotropic Elasticity of the Human Vocal Fold

Goodyer EN1*, Gunderson ML2, Tateya C3, Tateya T4,5 and Dailey SH6
1De Montfort University, Bio-Informatics Research Group, UK
2University of Wisconsin Madison, School of Veterinary Medicine, USA
3Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Japan
4Institute for Virus Research, Kyoto University, Japan
5The Hakubi Center, Kyoto University, Japan
6University of Wisconsin School of Medicine and Public Health, Department of Surgery, Division of Otolaryngology-Head and Neck Surgery, USA
Corresponding author : Eric Goodyer
Professor of Instrumentation, The Faculty of Technology, De Montfort University, The Gateway, Leicester, LE1 9BH, UK
Tel:
44 (0) 1289 309784
E-mail: [email protected]
Received: March 03, 2016 Accepted: April 12, 2016 Published: April 17, 2016
Citation: Goodyer EN, Gunderson ML, Tateya C, Tateya T, Dailey SH (2016) The Anisotropic Elasticity of the Human Vocal Fold. J Otol Rhinol 5:3. doi:10.4172/2324-8785.1000275

Abstract

Objective: To verify the anisotropic nature of the vocal folds by reproducing an experiment led by Rholfs that measured vocal fold elasticity in the transverse and longitudinal directions. To present a physiological explanation of the measured phenomena using immunochemical results.

Methods: 6 cadaveric human excised larynges were hemi-sectioned in the mid-sagittal plane exposing the vocal folds, and orthogonal measurements of tension made at 3 equally spaced points. Immunohistochemistry carried out by Ichiro Tateya was used to visualise collagens and elastins in the deep layer of the lamina propria.

Results: The LSR results indicate that the measured elasticity of the vocal folds are highly anisotropic. The immunohistochemistry results show that there is a strong alignment of collagens and elastins along the longitudinal axis of human vocal folds.

Conclusion: The measured anisotropic behaviour is due to the alignment of collagens & elastins in the lamina propria.

Keywords: Elasticity; Vocal folds

Keywords

Elasticity; Vocal folds

Introduction

Understanding if vocal fold viscoelasticity is isotropic or anisotropic is essential, as it brings to bear on multiple evolving approaches related to restoring or reconstructing the vocal fold lamina propria. Mitchell & Tojeira present methodologies for the construction of augmentation materials using scaffolds which are inherently anisotropic [1], and the need for anisotropic structures are found in other fields, such as musculoskeletal tissue engineering [2], regenerative cardiac tissue engineering [3], and nerves [4]. As vocal folds are required to perform mechanically, it is reasonable to extend the need to deploy anisotropic engineered tissue to phonosurgery. We therefore undertook a rheologic evaluation of human cadaveric vocal folds to confirm the results presented by Rohlfs [5], which demonstrated that the vocal folds inherently exhibit an anisotropic nature using the Linear Skin Rheometer [6-9]; and present immunochemical results obtained by Tateya et al., which show that there is an alignment of elastins and collagens in the longitudinal axis of the vocal folds [10]

Materials and Methods

With University of Wisconsin School of Medicine and Public Health Institutional Review Board and the Ethics Board of De Montfort University (DMU) approval, six human larynges were used. Visual inspection revealed normal appearing vocal folds in 5 out of 6 larynges, obtained from autopsy cases, frozen within 24 hours after death using liquid nitrogen, and stored at -80ºC. The larynges were thawed overnight at 4ºC and gradually warmed to 37ºC. Each was sharply divided in the mid-sagittal plane into two hemi-larynges and affixed to a Styrofoam base with pins to prevent movement (Figure 1).
Figure 1: Typical experimental set up.
The LSR was attached using a 2mm internal diameter bore cannula, with a vacuum pressure of approximately 50 millibar. No epithelial violation or tear was noted. A cyclical shear force of 1g was applied in both the transverse and longitudinal directions, and the resultant displacements logged. Five readings were taken from the mid-membranous site in both directions and averaged, the intention being to limit the tissue under test to be the free edge of the LP layer. The units used for all shear modulus results are Pascals.
The linear skin rheometer
The LSR applies a sinusoidal force of ± 1 g tangentially to the tissue, thus applying a shear force. The resultant strain is measured. A linear regression is applied to the force and displacement cycles, from which we derive the Dynamic Spring Rate (DSR) in units of force applied to achieve a unit of linear displacement. DSR is also known as stiffness or rigidity, and is measured in units of g/mm.
The LSR applies a sinusoidal force of ± 1 g tangentially to the tissue, thus applying a shear force. The resultant strain is measured. A linear regression is applied to the force and displacement cycles, from which we derive the Dynamic Spring Rate (DSR) in units of force applied to achieve a unit of linear displacement. DSR is also known as stiffness or rigidity, and is measured in units of g/mm.
Immunohistochemistry for collagen and elastin
Vocal folds were cut with sharp blades into 10-15 mm thick sections, soaked in embedding medium (O.C.T. compound, Tissue- Tek, Kyoto, Japan), frozen quickly with a combination of acetone and dry ice, and kept at -80°C. The samples were sectioned coronally or horizontally. Frozen sections of 30 μm thickness were made from each sample, and mounted on slides (Surperfrost / Plus Microscope Slides, Fisher Scientific, Pittsburgh, PA), air-dried and stored at -20°C until use.
Collagen types I, III, and elastin were identified by immunohistochemistry. The slides were washed 3 times with PBS and fixed for 2 minutes at room temperature in 4% paraformaldehyde, followed by washing in PBS 3 times. After blocking for 2 hours at room temperature, sections were incubated in primary antibody solution overnight at 4°C. Working dilutions and sources of antibodies used in this study included mouse Monoclonal Anti-Collagen Type I Clone COL-1 (Sigma, Birmingham, AL) at 1:1000, mouse Monoclonal Anti-Collagen Type III Clone FH-7A (Sigma, Birmingham, AL) at 1:2000, and mouse Anti Elastin Monoclonal antibody (Chemicon, Temecula, CA) at 1:100. These anti collagen antibodies had no or negligible cross-reactivity with the other types of collagen.
Samples were washed and incubated for 2 hours at room temperature with the second antibody solution. TOTO-3 (200 nM, Molecular probes, Eugene, OR) was also mixed in this procedure for nuclear staining. Finally, samples were washed 3 times in PBS and mounted on coverslips in Vectashield (Vector Labs, Burlingame, CA) for observation under a Laser Scanning Confocal Imaging System (BIO-RAD MRC-1024, Hercules, CA). The images of serial optical sections were collected every 1.5 μm in depth for three-dimensional reconstruction. Omission of the primary antibody served as a negative control. Working dilutions and sources of second antibodies used in the immunofluorescence technique included Texas-Red conjugated anti-mouse IgG (Jackson immunoresearch, PA) diluted to 1:200, and Texas-red anti-rabbit IgG (Jackson immunoresearch, PA) diluted to 1:400. As a blocking solution, 5% normal goat serum in 0.1% Triton-X with PBS was used. One percent normal goat serum in 0.1% Triton-X with PBS was used in the primary and second antibody reactions.

Results

Biomechanical results
Table 1 shows the results for all six larynges being a mean ratio of 2.48, meaning that vocal fold tension in the longitudinal direction is 2.48 times higher than the transverse direction. The mean coefficient of variance for all the data used is 12%.
Table 1: Shear modulus taken in the Transverse & Longitudinal directions.
Confidence determination in the data was obtained by comparison of left side data versus right side data, resulting in a correlation coefficient (CC) of 0.86, where 1.0 is a perfect match.
The value of 0.86 is acceptable, but a larger sample needs to be taken to increase our confidence in the conclusion.
Immunohistochemistry results
The distribution patterns of collagen subtypes and elastin in vocal folds detected by immunofluorescence technique were the same as previously described by Tateya et al. [10]. In the deep layer of the lamina propria (DLLP), collagen type I, collagen type III and elastin constructed fibers and were well-organized in the direction vertical to the vocal fold edge of the coronal plane and parallel to the vocal fold edge of the horizontal plane. Representative images are shown in Figure 2 and scale bars are 10 μm.
Figure 2A Collagen Type 1 48 year old Female 60x: This is the DLLP of the vocal fold sectioned parallel to the vocal fold edge of the horizontal plane. Magenta is collagen type I and green is nuclei. Type I fibers were thinner than most of type III fibers
Figure 2B Collagen Type 3 48 year old female x60x: This is the DLLP of the vocal fold sectioned parallel to the vocal fold edge of the horizontal plane. Magenta is collagen type III and green is nuclei. There were various diameters of collagen type III fibers. Most of them were thick and wavy and some were thin.
Figure 2C Collagens type 1 & 3 55 year old male 120x: This is the DLLP of the vocal fold sectioned vertical to the vocal fold edge of the coronal plane. Magenta is collagen type I and green is collagen type III.
Figure 2D Elastin 48 year old female 60x: This is the DLLP of the vocal fold sectioned parallel to the vocal fold edge of the horizontal plane. Magenta is elastin and green is nuclei. Elastin fibers were less wavy than collagen type I and type III.
Figure 2E Elastin and Collagen Type 3 55 year old male 120x: This is the DLLP of the vocal fold sectioned vertical to the vocal fold edge of the coronal plane. Magenta is elastin and green is collagen type III.
Figure 2: Stains showing Collagen and Elastin Alignments in the Deep Layer of the Lamina Propria. Figures A,B,D are 60 times magnification. Figures C and E are 120 times magnification. The scale bar is 10 μm.

Discussion

Quantifying the extent of the anisotropic nature of vocal folds has only been recently presented in the published literature. This is due to the nature of available apparatus, which is often based on techniques such as parallel plate rheometry that cannot resolve out strain with respect to the direction of applied stress. The LSR has enabled measurement of the stiffness of the LP to be measured without the need to dissect it our its anatomical context, and the because of the linear nature of the applied force it is now possible to be obtain strain data with respect to the direction of the applied stress. The motivation for this study was to confirm the findings of the team led by Rohlfs that the vocal folds present a highly anisotropic nature, and to determine if the cause of the measurable anisotropic behaviour of the LP is due to the alignment of collagens and elastins.
This was achieved by bringing together the methods used in two previous unrelated studies, into the biomechanical and the immunohistochemical properties of the vocal folds. By repeating and fusing these studies we conclude that the vocal folds are inherently anisotropic, and the underlying cause being that the collagens and elastins have a strong directional alignment, as presented in Figures 2A-2E.

Acknowledgments

This work was carried out with the support of the Engineering Physics and Science Research Council (EPSRC) of the United Kingdom

Conflicts of Interest

None

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