Research Article| Volume 37, ISSUE 3, P348-354, May 2023

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Hydration State and Hyaluronidase Treatment Significantly Affect Porcine Vocal Fold Biomechanics

Published:February 01, 2021DOI:



      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.


      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.


      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).


      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.

      Key Words

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      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

        • 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
        • Witt RE
        • Taylor LN
        • Regner MF.
        B.S. and JJJ. Effects of surface dehydration on mucosal wave amplitude and frequency in excised canine larynges.
        Otolaryngol Head Neck Surg. 2011; 144: 108-113
        • 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
        • 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
        • Min Y
        • Lemke J
        • Mersbergen M
        • et al.
        Biological mechanisms underlying voice changes due to dehydration.
        J Speech Lang Hear Res. 2002; 4388
        • Verdolini-marston K
        • Titze R
        • Druker DG.
        Changes in phonation threshold pressure with induced conditions of hydration.
        J Voice. 1990; 4: 142-151
        • 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
        • Verdolini K.
        Dependence of phonatory effort on hydration level.
        J Speech Hear Res. 1994; 37: 1001-1007
        • 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
        • 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
        • 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
        • Zhang Z.
        Mechanics of human voice production and control.
        J Acoust Soc Am. 2017; 2614
        • Ward PD
        • Thibeault SL
        • Gray SD.
        Hyaluronic acid: its role in voice.
        J Voice. 2002; 16: 303-309
        • 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
        • 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
        • Gaston J
        • Thibeault SL.
        Hyaluronic acid hydrogels for vocal fold wound healing.
        BioMatter. 2013; 3: e23799
        • Chhetri DK
        • Mendelsohn AH.
        Hyaluronic acid for the treatment of vocal fold scars.
        Curr Opin Otolaryngol Head Neck Surg. 2010; 18: 498-502
        • 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;
        • Cooper DS.
        Elasticity of canine vocal fold tissue.
        J Speech Hear Res. 1984; 27: 212-219
        • Dinesh K
        • Chhetri
        Young's modulus of canine vocal fold cover layers.
        J Voice. 2015; 28 (Young): 406-410
        • Alipour F.
        Vocal fold elasticity in the pig, sheep and cow larynges.
        J Voice. 2012; 25 (Vocal): 130-136
      2. 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.

        • Jiang JJ.
        Comparison of the phonation-related structures among pig, dog, white-tailed deer, and human larynges.
        Ann Otol Rhinol Laryngol. 2001; : 1120-1125
        • Alipour F
        • Jaiswal S.
        Glottal airflow resistance in excised pig, sheep, and cow larynges.
        J Voice. 2009; 23: 40-50
        • Alipour F
        • Jaiswal S.
        Phonatory characteristics of excised pig, sheep, and cow larynges.
        J Acoust Soc Am. 2008; 123: 4572-4581
        • Woodson G.
        Developing a porcine model for study of vocal fold scar.
        J Voice. 2012; 26: 706-710
        • 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
        • 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; (Functional): 1-28
        • 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
        • Haraldsson BT
        • Aagaard P
        • Krogsgaard M
        • et al.
        Region-specific mechanical properties of the human patella tendon.
        J Appl Physiol. 2005; 98: 1006-1012
        • 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
        • Schindelin J
        • Arganda-Carrera I
        • Frise E
        • et al.
        Fiji - an Open platform for biological image analysis.
        Nat Methods. 2009; 9 (Fiji)
        • 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
        • Burks G
        • de Vita R
        • Leonessa A.
        Characterization of the continuous elastic parameters of porcine vocal folds.
        J Voice. 2020; 34: 1-8
        • 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
        • Fisher K V
        Vocal fold surface hydration: a review.
        J Voice. 2010; 23 (Vocal): 658-665
        • Zhang Y
        • Czerwonka L
        • Tao C
        • et al.
        A biphasic theory for the viscoelastic behaviors of vocal fold lamina propria in stress relaxation.
        J Acoust Soc Am. 2015; 1627
        • 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
        • 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
        • Chan RW
        • Gray SD
        • Titze IR.
        The importance of hyaluronic acid in vocal fold biomechanics.
        Otolaryngol Head Neck Surg. 2001; 124: 607-614
        • 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