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. Author manuscript; available in PMC: 2024 May 1.
Published in final edited form as: J Voice. 2021 Feb 2;37(3):348–354. doi: 10.1016/j.jvoice.2021.01.014

Hydration State and Hyaluronidase Treatment Significantly Affect Porcine Vocal Fold Biomechanics

Chenwei Duan *,†, Julian M Jimenez *,‡, Craig Goergen *, Abigail Cox §, Preeti M Sivasankar *,†, Sarah Calve *,‡
PMCID: PMC8325720  NIHMSID: NIHMS1669140  PMID: 33541766

Summary:

Objectives.

The understanding of vocal fold hydration state, including dehydrated, euhydrated, rehydrated tissue, and how hydration affects vocal fold biomechanical properties is still evolving. Although clinical observations support the benefits of increasing vocal fold hydration after dehydrating events, more mechanistic information on the effects of vocal fold dehydration and the beneficial effects of rehydration are needed. Alterations to hyaluronic acid (HA), an important component of the vocal fold extracellular matrix, are likely to influence the biomechanical properties of vocal folds. In this study, we investigated the influence of hydration state and HA on vocal fold tissue stiffness via biomechanical testing.

Study design.

Prospective, ex vivo study design.

Methods.

Fresh porcine vocal folds (N = 18) were examined following sequential immersion in hypertonic (dehydration) and isotonic solutions (rehydration). In a separate experiment, vocal folds were incubated in hyaluronidase (Hyal) to remove HA. Control tissues were not exposed to any challenges. A custom micromechanical system with a microforce sensing probe was used to measure the force-displacement response. Optical strain was calculated, and ultrasound imaging was used to measure tissue cross-sectional area to obtain stress-strain curves.

Results.

Significant increases (P ≤ 0.05) were found in tangent moduli between dehydrated and rehydrated vocal folds at strains of ε = 0.15. The tangent moduli of Hyal-digested tissues significantly increased at both ε = 0.15 and 0.3 (P ≤ 0.05).

Conclusion.

Vocal fold dehydration increased tissue stiffness and rehydration reduced the stiffness. Loss of HA increased vocal fold stiffness, suggesting a potential mechanical role for HA in euhydrated vocal folds.

Keywords: Biomechanics, Dehydration, Hyaluronic acid, Rehydration, Vocal folds

INTRODUCTION

Clinically, hydration treatments are hypothesized to maintain the viscous properties of vocal folds, which is necessary for efficient voice production.1 However, our understanding of the relationship between vocal fold tissue hydration level and biomechanical properties is still evolving. Ex vivo experiments demonstrated that dehydration altered vocal fold oscillatory properties.2,3 The relationship between hydration and vocal function is further supported by human subject research.4,5 Previous research demonstrated that dehydrated vocal folds need higher phonation threshold pressure (the minimum lung pressure required to initiate and sustain vocal fold oscillation).6 Rehydration is associated with improvements in voice quality (decreased shimmer, jitter, phonation threshold pressure, and increased maximum phonation time and maximum fundamental frequency values).7–9. Despite these correlative clinical observations, the benefits of increased vocal fold hydration from a tissue mechanics aspect remain unclear. Increase in the stiffness of vocal folds is generally hypothesized to impact tissue vibration and resulting voice production.10,11 For example, increased stiffness significantly increased the fundamental frequency, along the anterior-posterior axis.12 Therefore, we choose to use stiffness as an index in this study to investigate the biomechanical influence of hydration state on vocal folds via uniaxial tensile testing.

The biomechanical properties of vocal folds are dictated by the extracellular matrix (ECM) of the lamina propria.13 Hyaluronic acid (HA) is an important component of the ECM and influences the mechanical response of vocal folds by keeping the tissue hydrated through the strong affinity of water to negatively charged HA chains.14 Native and functionalized derivatives of HA have received extensive attention as injection agents for the treatment of vocal fold scarring and to help restore vocal function.15–17 A previous study showed that injured vocal folds treated with the HA derivative Extracel™ had significantly improved biomechanical properties compared with saline-treated controls.16 Despite reports of the importance of HA in the vocal fold and its role in influencing hydration state,18 how HA directly affects the biomechanical properties of the vocal fold has yet to be demonstrated. This is an important question because HA quantity is reduced in systemically dehydrated rats which could potentially underlie the change in mechanical behavior of dehydrated vocal folds.19 Therefore, we quantified in this study the effects of HA removal on vocal fold stiffness.

Excised larynges of various species have been used to study mechanical properties of the vocal folds.20–22 The ECM architecture of the pig (porcine) vocal fold has a similar arrangement to that found in the human.23 Several studies have also emphasized the benefits of using a porcine animal model in studies of normal physiology of phonation.24–26 and in vocal fold injury.27 Therefore, porcine larynges were utilized to study how the mechanical properties of vocal folds vary in response to different hydration states.

Our previous studies demonstrated a decrease in HA after dehydration.19,28 To follow up on the potential change in biomechanical behavior after dehydration in this study, we hypothesized that the optimal stiffness of vocal folds would be impacted after dehydration via losing both water and HA, but that the stiffness properties would recover through rehydration. To test this hypothesis, we experimentally treated samples using two different approaches: (1) porcine vocal folds were immersed in hypertonic solution (15% NaCl in ddH2O) and phosphate-buffered saline (PBS) sequentially to mimic dehydration and rehydration, and (2) porcine vocal folds were incubated with hyaluronidase (Hyal) to mimic HA loss during dehydration. We determined the stiffness of vocal folds by measuring the stress-strain response during loading at physiologic levels.

In the present study, several optimizations were made to address some of the limitations inherent to mechanical testing.29,30 A custom micromechanical system was employed to determine the force-displacement response of excised vocal fold tissues. Grip-to-grip measurement displacement may underestimate tissue mechanical properties because strain is concentrated at the grip interface.31 However, optical measurement of tissue strain provides a better understanding for tissue region-specific mechanical property.32 Therefore, a dissecting microscope with a camera was used to acquire videos of the tissue during loading to enable optical strain calculations. To convert force to stress, ultrasound imaging of tissue ex vivo was exploited to measure the cross-sectional area of vocal fold tissue. The stress-strain response was then plotted to determine how vocal fold stiffness changes in response to Hyal treatment and dehydration. Overall, these findings provide insight into how hydration status and ECM composition influence vocal fold mechanical properties.

METHODS

Sample collection and preparation

Fresh healthy porcine larynges (N = 9) were obtained from Monon Meat Packing Inc, an Indiana state-inspected and approved abattoir, and transported on ice. Following the removal of pharyngeal and extrinsic laryngeal muscles to allow access to the laryngeal cartilaginous framework, each larynx was bisected along the midsagittal plane to create two hemi-larynges. The true vocal fold, including epithelium and lamina propria, was dissected from the hemilarynx. The vocalis muscle was not included in the sample. Next, the vocal fold was secured with Ultra Gel Control Super Glue (Loctite, Westlake, OH) to a custom laser cut polyethylene terephthalate frame (Figure 1A), then immediately immersed in PBS to prevent both the tissue from drying out and the glue from spreading onto the midportion of the sample. The whole process from obtaining to mounting the vocal folds took place no more than 2–3 hours postmortem.

FIGURE 1.

FIGURE 1.

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Representative image of porcine vocal fold sample secured with glue on a polyethylene terephthalate (PET) frame before (A) and after (B) addition of fiducial lines.

Fiducial lines on tissue surface

Before conducting tensile tests, tissue marking dye (Thermo Fisher Scientific, Waltham, MA) was used to create fiducial lines on the surface of the tissue. Four fiducial lines were marked on each sample by using pipette tips to separate the whole tissue into three equal parts (Figure 1B).

Mechanical test setting

A custom 3D-printed spring (Figure 2) and a commercially available micromanipulator system (FT-RS1002, Femtotools, Zürich, Switzerland), outfitted with a microforce sensing probe (FT-S100’000, Femtotools), were used to perform uniaxial tensile tests on the tissues. The micromanipulator was used to stretch vocal folds to a specific strain at a specified strain rate while the microforce sensor acquired force data. The sample was placed on the 3D-printed spring. We attached a hook on the 3D-printed platform to the microforce sensing probe to keep the electronics of the probe away from the aqueous solutions (Figure 3). Each sample was fully submerged during tensile testing. A dissecting microscope (Leica M80) with a camera (Lumenera INFIN-ITY3–3URC) recorded the loading and unloading process at 5 frames per second for each sample throughout the experiment from which strain was calculated. A schematic of the test apparatus is shown in Figure 4.

FIGURE 2.

FIGURE 2.

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Representative image showing 3D-printed spring dimensions. All dimensions in mm. Schematic of an isometric view of the spring (A). A top view of the spring and sample chamber (B).

FIGURE 3.

FIGURE 3.

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Representative image of the setup of 3D-printed spring, microforce sensor, and porcine vocal fold sample. The connection between 3D printed spring and microforce sensor (A). A SolidWorks schematic of 3D-printed spring, microforce sensor, porcine vocal fold sample, frame, and aqueous solution (B).

FIGURE 4.

FIGURE 4.

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Dissecting microscope and mechanical testing apparatus. The spring, sensor, and micromanipulator are labeled in the image.

Dehydration and rehydration protocol

Right and left porcine vocal folds (N = 8) were randomly assigned to a dehydrated group and a sham control group. For dehydration, each vocal fold was first submerged in a hypertonic solution (15% NaCl in ddH2O) at room temperature for 30 minutes. The tissues were then stretched to ε = 0.5 and ε = 0.7 in hypertonic solution, at a strain rate of 0.01 s−1. To simulate rehydration recovery, each sample was immersed in isotonic solution (PBS) at room temperature for 30 minutes and then stretched to ε = 0.5 and ε = 0.7 in PBS, at a strain rate of 0.01 s−1. Tissue in the sham control group were immersed in PBS and tested to the same strains and strain rates as the dehydrated and rehydrated tissues.

Hyaluronidase digestion protocol

Ten porcine vocal folds were randomly divided into hyaluronidase digestion (Hyal-digested) and control groups. In the Hyal-digested group, the porcine vocal folds were incubated in 2 mg/mL hyaluronidase (Sigma-Aldrich, St. Louis, MO), dissolved in PBS at 37°C, and put in a thermomixer shaker with incubator block (Eppendorf, Hamburg, Germany) at 300 rpm for 2 hours. Following a brief wash with PBS, the porcine vocal folds were submerged in PBS and stretched to ε = 0.5 and ε = 0.7, both at a strain rate of 0.01 s−1. The vocal folds in the control group were always immersed in PBS, shaken at 300 rpm for 2 hours at 37°C, and stretched to ε = 0.5 and ε = 0.7, both at a strain rate of 0.01 s−1.

Ultrasound imaging setting

To accurately quantify the mechanical properties of the vocal folds, we converted the force-displacement responses into stress-strain curves. To achieve the conversion, a Vevo 3100 high frequency ultrasound imaging system (FUJIFILM VisualSonics, Toronto, Canada) was used to measure the cross-sectional area of the vocal folds before each experiment. We collected 3D images of the vocal fold sample by scanning along the longitudinal axis using a linear step motor and an MX700 transducer with a 0.08 mm step size (29–71 MHz), producing datasets with an axial resolution of 30 μm. Images were analyzed in Vevo LAB software (FUJIFILM VisualSonics). The boundary of the vocal fold sample was manually segmented and the generated 2D area was recorded at multiple locations (10–15 spots) and then averaged to produce a representative cross-sectional area.

Optical strain calculation

The video of vocal fold loading and unloading was converted to individual image frames in ImageJ using virtual stack. A customized macro was developed in FIJI (NIH) to get the X and Y coordinates for each fiducial line throughout the loading and unloading cycle.33 The manually drawn polyline X and Y coordinates files were then analyzed through a customized MATLAB (MathWorks, Natick, MA) algorithm in MATLAB_R2020a (version 9.8.0). Briefly, one reference image of the nominal undeformed sample was chosen as the first file in sequence to load. For each frame, differences of X values indexed by Y values for the fiducial lines were calculated. The average distance between each pair of lines was used to calculate strain.

Statistical analysis

All statistical analyses were completed with GraphPad Prism software (version 8.4.0). Tangent moduli of Hyal-digested and control groups were compared using two-way ANOVA followed by multiple comparisons. Tangent moduli of dehydration and rehydration groups were compared using paired t tests. Likewise, tangent moduli of control tissue at timepoints matched for dehydration and rehydration were compared using paired t tests. Significance was set to P ≤ 0.05 for all statistical analyses.

RESULTS

Tangent moduli of porcine vocal folds in response to dehydration and rehydration

There were significant differences in tangent moduli at ε = 0.3 (P = 0.02) between dehydrated and sham control vocal folds. With posthoc testing, significant differences in tangent moduli were obtained between dehydrated and rehydrated vocal folds at ε = 0.15 (P = 0.04) but not ε = 0.3. For the sham control group, no significant differences in tangent moduli were observed at either at ε = 0.15 or ε = 0.3. Figure 5A summarizes tangent moduli at ε = 0.15 and ε = 0.3 of vocal folds in the dehydrated, rehydrated, and sham control groups. Figure 5B shows the stress-strain curves of vocal folds in the dehydrated, rehydrated, and sham control groups.

FIGURE 5.

FIGURE 5.

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Bar graph (Mean, SEM) of the tangent moduli at ε = 0.15 and ε = 0.3 of porcine vocal fold in dehydration, rehydration and sham control group (N = 4/group; A). Significant differences in tangent moduli were found between dehydrated and rehydrated vocal folds at ε = 0.15 but not ε = 0.3. No significant differences in tangent moduli were found in sham control group. *: P ≤ 0.05. Representative stress-strain curves of porcine vocal fold in dehydration, rehydration, and sham control group (B).

Tangent moduli of porcine vocal folds in Hyal-digested and control groups

There were significant differences in tangent moduli at ε = 0.15 (P = 0.0312) and ε = 0.3 (P = 0.0001) between Hyal-digested and control groups. After hyaluronidase incubation, loss of HA increased the stiffness of the vocal folds when compared with the control group. Figure 6A summarizes tangent moduli at ε = 0.15 and ε = 0.3 of vocal folds in the Hyal-digested and the control group. Figure 6B shows the stress-strain curves of vocal folds in Hyal-digested and the control group during tensile tests.

FIGURE 6.

FIGURE 6.

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Bar graph (Mean, SEM) of the tangent moduli at ε = 0.15 and ε = 0.3 of porcine vocal fold in Hyal-digested and control group (N = 5/group; A). Hyaluronidase incubation of the vocal folds significantly increased the tangent moduli when compared with the control group. *: P ≤ 0.05. Representative stress-strain curves of porcine vocal fold in Hyal-digested and control group (B).

DISCUSSION

This study investigated the effects of dehydration, rehydration, and the influence of HA removal on the mechanical properties of vocal fold tissue. Our results regarding vocal fold dehydration and rehydration induced through sequential immersion in hypertonic and isotonic solution showed an increase in stiffness in dehydrated vocal fold, and a decreasing trend in stiffness after rehydration. Our results also revealed that loss of HA increased the stiffness of pig vocal fold.

In this study, the tangent moduli in the control group were measured at ε = 0.15 (5.9 ± 1.1 kPa) and ε = 0.3 (14.3 ± 3.5 kPa), as strain values for the pig vocal folds are reported to be between ~16% and ~29% along the anterior-posterior axis during phonation.34 Previous studies reported that the Young’s modulus of normal pig superior vocal fold was 19.2 ± 4.2 kPa22 and elastic parameters of pig inferior vocal fold were found to be in the range of 17.86 kPa and 609.27 kPa35. While the data in the current study differ from published literature, the reasons for this could be the contrast between fresh pig vocal fold tissues in this study compared to frozen and rethawed porcine vocal folds in previous work.22,35 In other previous work, freezing tissues have been shown to change the material properties for articular cartilage.36 Another potential reason was that in this study strain was calculated based on optical markers, as opposed to actuator displacement, which can underestimate stiffness due to localized deformation when testing soft tissues. A major advantage of using optical displacement to calculate strain, as opposed to the displacement of the micromanipulator, is that the deformation of the pig vocal fold is observed directly, eliminating potential errors attributed to the pig vocal fold slipping from the glue that was used to adhere this tissue to the polyethylene terephthalate frame, or potential deformation of the spring during tensile tests. Additionally, a custom 3D-printed spring configuration enabled (1) the specimens to remain hydrated while keeping the electronics of the probe away from the aqueous solution and (2) the integration of the microforce sensor and optical marker visualization during uniaxial tensile tests.

The water content in vocal folds is likely important for facilitating cellular interactions, maintaining optimal biomechanical characteristics, and ensuring normal voice quality.37 Previous clinical studies showed that increased vocal fold hydration level can improve voice quality,7,9 and benefits patients with laryngeal nodules or polyps.8 In this study, we quantified the effects of dehydration and rehydration on the biomechanical properties of porcine vocal folds (Figure 5). The results demonstrated that stiffness of vocal fold increased after water was expelled by osmotic pressure. By rehydrating with PBS, vocal fold stiffness recovered to some extent when compared with the dehydrated status. The results suggest that dehydration caused a disruption in the organization and physical support between ECM components, making it difficult to completely recover simply through PBS immersion. Our results are consistent with the biphasic equations developed by Zhang et al,38 which suggested that removing water from tissue induced a collapse of the solid structure and a decrease in pore size.

HA contributes to optimal vocal fold vibration and prevents the tissue from oscillatory trauma during voice production.17 However, a previous study showed that significant voice improvements were found after injection of hyaluronidases on patients with vocal hemorrhage and Reinke edema,39 suggesting that optimal amount of HA was important for maintaining voice quality. To test the effect of HA on the biomechanical properties of porcine vocal folds, tissues were incubated with hyaluronidase and we found the loss of HA significantly increased the stiffness (Figure 6). Our results are comparable with several other studies in bovine cartilage36 and pancreatic islets40 that also showed reducing the amount of HA using Hyal increased tissue stiffness. Notably, Chan et al found that removal of HA from human vocal fold resulted in a decrease in the shear storage modulus and an increase in dynamic viscosity.41 These contrasting results are likely due to differences in tissue source, sample storage and treatment, and testing modality. Fresh porcine tissues were uniaxially tensile tested in this study, whereas previously frozen tissue was tested dynamically using a rheometer in the human vocal fold study. Although our results support the idea that HA influences the mechanical properties of pig vocal fold tissue, it is important to note that Hyal mainly hydrolyzes β-N-acetyl hexosamine glycosidic bonds in HA, but chondroitin sulfate sidechains of proteoglycans in the ECM can still be cleaved to some extent.42 Future studies will tease apart the role of various proteoglycans in vocal fold tissue.

CONCLUSIONS

In this study, we investigated changes in biomechanical behavior of excised porcine vocal fold following dehydration with two approaches: (1) sequentially immerse in hypertonic and isotonic solution to mimic dehydration and rehydration, and (2) challenge with hyaluronidase to remove HA. To measure biomechanical outcomes a micromanipulator system and a customized spring were used to conduct tensile tests, together with an optimized method of stress-strain calculation. Our results showed an increase in stiffness in dehydrated vocal fold, and a decreasing trend in stiffness after rehydration. In addition, loss of HA significantly increased vocal fold stiffness. Combining the results together, this study demonstrated that dehydration impacted vocal fold biomechanical properties through both loss of water and disturbance of HA. The potential for dehydration to disrupt the ultrastructure of ECM and alter the mechanical properties of the vocal folds necessitates further research.

Funding:

Funding was provided from R01DC0115545 (National Institutes of Health/National Institute on Deafness and other Communication Disorders) and NSF CMMI 1911346 (National Science Foundation/Division of Civil, Mechanical & Manufacturing Innovation).

Footnotes

Conflict of Interest: None to report.

REFERENCES

  • 1.Franca MC, Simpson KO. Effects of hydration on voice acoustics. Contemp Issues Commun Sci Disord. 2009;36(Fall):142–148. doi: 10.1044/cicsd_36_f_142. [DOI] [Google Scholar]
  • 2.Witt RE, Regner MF, Tao C, et al. The effect of dehydration on phonation threshold flow in excised canine larynges. Ann Otol Rhinol Laryngol. 2009;118:154–159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Witt RE, Taylor LN, Regner MF. S. B and J JJ. Effects of surface dehydration on mucosal wave amplitude and frequency in excised canine larynges. Otolaryngol Head Neck Surg. 2011;144:108–113. 10.1177/0194599810390893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Alves M, Krüger E, Pillay B, et al. The effect of hydration on voice quality in adults: a systematic review. J Voice. 2019;33:125.e13–125. e28. 10.1016/j.jvoice.2017.10.001. [DOI] [PubMed] [Google Scholar]
  • 5.van Wyk L, Cloete M, Hattingh D, et al. The effect of hydration on the voice quality of future professional vocal performers. J Voice. 2017;31:111.e29–111.e36. 10.1016/j.jvoice.2016.01.002. [DOI] [PubMed] [Google Scholar]
  • 6.Min Y, Lemke J, Mersbergen M, et al. Biological mechanisms underlying voice changes due to dehydration. J Speech Lang Hear Res. 2002;4388. 10.1044/1092-4388(2002/021. [DOI] [PubMed] [Google Scholar]
  • 7.Verdolini-marston K, Titze R, Druker DG. Changes in phonation threshold pressure with induced conditions of hydration. J Voice. 1990;4:142–151. [Google Scholar]
  • 8.Verdolini-marston K, Sandage M, Titze IR. Effect of hydration treatments on laryngeal nodules and polyps and related voice measures. J Voice. 1994;8:30–47. [DOI] [PubMed] [Google Scholar]
  • 9.Verdolini K Dependence of phonatory effort on hydration level. J Speech Hear Res. 1994;37:1001–1007. 10.1044/jshr.3705.1001. [DOI] [PubMed] [Google Scholar]
  • 10.Zhang Z, Hieu Luu T. Asymmetric vibration in a two-layer vocal fold model with left-right stiffness asymmetry: experiment and simulation. J Acoust Soc Am. 2012;132:1626–1635. 10.1121/1.4739437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zhang Z Effect of vocal fold stiffness on voice production in a three-dimensional body-cover phonation model. J Acoust Soc Am. 2017;142:2311–2321. 10.1121/1.5008497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Zhang Z Cause-effect relationship between vocal fold physiology and voice production in a three-dimensional phonation model. J Acoust Soc Am. 2016;139:1493–1507. 10.1121/1.4944754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhang Z Mechanics of human voice production and control. J Acoust Soc Am. 2017;2614. 10.1121/1.4964509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ward PD, Thibeault SL, Gray SD. Hyaluronic acid: its role in voice. J Voice. 2002;16:303–309. 10.1016/S0892-1997(02)00101-7. [DOI] [PubMed] [Google Scholar]
  • 15.Finck CL, Harmegnies B, Remacle A, Lefebvre P. Implantation of esterified hyaluronic acid in microdissected Reinke’s space after vocal fold microsurgery: short- and long-term results. J Voice. 2010;24:626–635. 10.1016/j.jvoice.2008.12.015. [DOI] [PubMed] [Google Scholar]
  • 16.Thibeault SL, Klemuk SA, Chen X, et al. In vivo engineering of the vocal fold ECM with injectable HA hydrogels - Late effects on tissue repair and biomechanics in a rabbit model. J Voice. 2011;25:249–253. 10.1016/j.jvoice.2009.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gaston J, Thibeault SL. Hyaluronic acid hydrogels for vocal fold wound healing. BioMatter. 2013;3:e23799. http://www.landesbio-science.com/journals/biomatter/2012BIOMATTER0047R.pdf%5Cnhttp://ovidsp.ovid.com/ovidweb.cgi?T=JS&PAGE=reference&D=emed15&NEWS=N&AN=368566776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Chhetri DK, Mendelsohn AH. Hyaluronic acid for the treatment of vocal fold scars. Curr Opin Otolaryngol Head Neck Surg. 2010;18:498–502. 10.1097/MOO.0b013e32833f85d1. [DOI] [PubMed] [Google Scholar]
  • 19.Cox A, do Nascimento N, dos Santos A, et al. Dehydration and estrous staging in the rat larynx : an in vivo prospective investigation. J Voice. 2019. 10.1016/j.jvoice.2019.06.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Cooper DS. Elasticity of canine vocal fold tissue. J Speech Hear Res. 1984;27(May 2016):212–219. 10.1044/jshr.2702.212. [DOI] [PubMed] [Google Scholar]
  • 21.Chhetri Dinesh K. Young’s modulus of canine vocal fold cover layers. J Voice. 2015;28:406–410. 10.1016/j.jvoice.2013.12.003. Young. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Alipour F Vocal fold elasticity in the pig, sheep and cow larynges. J Voice. 2012;25:130–136. 10.1016/j.jvoice.2009.09.002. Vocal. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Garrett CG, Coleman JR, Reinisch L. Comparative histology and vibration of the vocal folds : implications for experimental studies in microlaryngeal surgery. Laryngoscope. 110:814–824. [DOI] [PubMed] [Google Scholar]
  • 24.Jiang JJ. Comparison of the phonation-related structures among pig, dog, white-tailed deer, and human larynges. Ann Otol Rhinol Laryngol. 2001:1120–1125. [DOI] [PubMed] [Google Scholar]
  • 25.Alipour F, Jaiswal S. Glottal airflow resistance in excised pig, sheep, and cow larynges. J Voice. 2009;23:40–50. 10.1016/j.jvoice.2007.03.007. [DOI] [PubMed] [Google Scholar]
  • 26.Alipour F, Jaiswal S. Phonatory characteristics of excised pig, sheep, and cow larynges. J Acoust Soc Am. 2008;123:4572–4581. 10.1121/1.2908289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Woodson G Developing a porcine model for study of vocal fold scar. J Voice. 2012;26:706–710. 10.1016/j.jvoice.2012.03.003. [DOI] [PubMed] [Google Scholar]
  • 28.Duan C, do Nascimento NC, Calve S, Sivasankar MP, et al. Restricted water intake adversely affects rat vocal fold biology. Laryngoscope. 2020;2:1–7. 10.1002/lary.28881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Sobieraj M, Amin MR, Branski RC. Functional assessment of the ex vivo vocal folds through biomechanical testing: A review. Mater Sci Eng C Mater Biol Appl. 2017:1–28. 10.1016/j.msec.2016.04.018. Functional. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Wu JZ, Brumfield A, Miller GR, Metheny R, Cutlip RG. Comparison of mechanical properties of rat tibialis anterior tendon evaluated using two different approaches. Bio-Med Mater Eng. 2004;14:13–22. [PubMed] [Google Scholar]
  • 31.Haraldsson BT, Aagaard P, Krogsgaard M, et al. Region-specific mechanical properties of the human patella tendon. J Appl Physiol. 2005;98:1006–1012. 10.1152/japplphysiol.00482.2004. [DOI] [PubMed] [Google Scholar]
  • 32.Arruda EM, Calve S, Dennis RG, et al. Regional variation of tibialis anterior tendon mechanics is lost following denervation. J Appl Physiol. 2006;101:1113–1117. 10.1152/japplphysiol.00612.2005. [DOI] [PubMed] [Google Scholar]
  • 33.Schindelin J, Arganda-Carrera I, Frise E, et al. Fiji - an Open platform for biological image analysis. Nat Methods. 2009;9. 10.1038/nmeth.2019. Fiji. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Bakhshaee H, Young J, Yang JCW, et al. Determination of strain field on the superior surface of excised larynx vocal folds using DIC. J Voice. 2013;27. 10.1038/jid.2014.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Burks G, de Vita R, Leonessa A. Characterization of the continuous elastic parameters of porcine vocal folds. J Voice. 2020;34:1–8. 10.1016/j.jvoice.2018.09.007. [DOI] [PubMed] [Google Scholar]
  • 36.Xu X, Li Z, Cai L, et al. Mapping the nonreciprocal micromechanics of individual cells and the surrounding matrix within living tissues. Nat Publishing Group. 2016:1–9. 10.1038/srep24272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Fisher KV Vocal fold surface hydration: a review. J Voice. 2010;23 (6):658–665. 10.1016/j.jvoice.2008.03.010. Vocal. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Zhang Y, Czerwonka L, Tao C, et al. A biphasic theory for the visco-elastic behaviors of vocal fold lamina propria in stress relaxation. J Acoust Soc Am. 2015;1627. 10.1121/1.2831739. [DOI] [PubMed] [Google Scholar]
  • 39.Woo P Hyaluronidase injection in the vocal folds for vocal hemorrhage, Reinke Edema, and hyaluronic acid overinjection: a novel application in the Larynx. J Voice. 2018;32:492–498. 10.1016/j.jvoice.2017.07.004. [DOI] [PubMed] [Google Scholar]
  • 40.Nagy N, de La Zerda A, Kaber G, et al. Hyaluronan content governs tissue stiffness in pancreatic islet inflammation. J Biol Chem. 2018;293:567–578. 10.1074/jbc.RA117.000148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Chan RW, Gray SD, Titze IR. The importance of hyaluronic acid in vocal fold biomechanics. Otolaryngol Head Neck Surg. 2001;124:607–614. 10.1067/mhn.2001.115906. [DOI] [PubMed] [Google Scholar]
  • 42.Sugahara K, Mikami T, Uyama T, et al. Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Curr Opin Struct Biol. 2003;13:612–620. 10.1016/j.sbi.2003.09.011. [DOI] [PubMed] [Google Scholar]

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