Biomechanical measurements of vocal fold elasticity

Transcription

1 Page 1 of 5 Tissue Engineering & Modelling Biomechanical measurements of vocal fold elasticity DR Dembinski 1, L Oren 1*, E Gutmark 2, SM Khosla 1 Abstract Introduction Accurate characterization of the elastic properties of vocal fold tissue is important in phonosurgical correction of vocal fold pathology and development of physiologic phonation models. This paper examines the work to date attempting to characterize the elastic response of the vocal fold tissue, focusing on three commonly used biomechanical testing modalities in the field: longitudinal elongation, linear skin rheometry, and microindentation. It is hoped that a thorough review of current literature in the field will identify strengths and weaknesses associated with each testing technique and suggest directions for future work. Conclusion While much progress has been made in the characterization of vocal fold elasticity, there is still work to be done to make elasticity measurements practical for clinical applications. Introduction The sound produced by the vibratory process of the human vocal folds is largely dependent on the tissue s elastic properties. For example, voice properties such as pitch and acoustic intensity are determined (in part) by the tissue strain 1. Alteration of local elastic parameters in the vocal fold tissue often results in perceivable speech pathology, often requiring phonosurgical correction. Accurate characterization of the inherent elastic parameters of the vocal folds, particularly the Young s modulus and shear modulus of the tissue, may help *Corresponding author orenl@ucmail.uc.edu 1 University of Cincinnati, Department of Otolaryngology-Head and Neck Surgery, Cincinnati, Ohio 2 University of Cincinnati, Department of Aerospace Engineering, Cincinnati, Ohio refine surgical techniques used to repair vocal fold pathology and improve computer simulations of the phonation cycle. There have been several studies that aimed to characterize the elastic characteristics of vocal fold tissue using varying biomechanical methods. However, due to the non-linear softtissue characteristics of the tissue, the estimates of the tissue elasticity are inconsistent, with reported values varying over several orders of magnitude 2. Difficulties in accurate estimation of vocal fold elasticity arise from a variety of sources, including but not limited to, differences in tissue composition, testing modality, and specimen type and preparation. They can also stem from differences in the Young s modulus estimation technique. The elastic characteristics of the vocal folds are commonly studied using several models, including human, canine, porcine, and synthetic specimens. While human samples generate the most clinically relevant data, it is often difficult and expensive to procure fresh specimens, resulting in significant tissue decomposition that affects measured elasticity values. Canine specimens are the preferred animal model used in speech science because of their similarities, both anatomically and acoustically, with human specimens 3. They also allow for testing immediately postmortem, minimizing tissue decomposition and allowing for more accurate elasticity measurements. Porcine larynges have been indicated as valid experimental models because they vibrate at a frequency similar to that of human larynges and they are easily obtainable 4. However, variations in anatomy, including the presence of two sets of oscillating vocal folds, prevents direct comparison of these samples to their human counterparts 5. Synthetic models are also constructed, mostly out of silicone, based on available literature data regarding anatomical structure and tissue composition 6. Elastic characteristics of these models are often difficult to correlate to human samples because of their inorganic composition. The following work presents a review of the data available in the literature regarding vocal fold elasticity. The data was primary collected using three biomechanical techniques: longitudinal elongation, linear skin rheometry, and microindentation. Each one of these methods is subsequently described and major findings from each study are tabulated and summarized. Longitudinal elongation The first attempt to characterize the elastic properties of vocal fold tissue was made by Ishizaka and Kaneko 7 who used the longitudinal elongation technique (longitudinal elongation results are summarized in table 1). In the traditional longitudinal elongation test, the tissue of the vocal fold is excised from the larynx and is fixed to the testing apparatus at its anterior/ posterior ends. The tissue is then subjected to a controlled, stepwise tension, and the corresponding forces are recorded. The tissue can be kept more viable by suspension in an aerated solution, such as a Krebs- Ringer solution. Cyclic stress-relaxation testing allows for generation of hysteresis curves, from which the stiffness of the tissue, which is measured by its Young s modulus, can be extracted. Ishizaka and Kaneko reported a constant stiffness value of 3.7kPa for a sample of the human vocal fold 7. Perlman et al. 8 isolated canine vocal fold specimens and used longitudinal elongation to determine elastic parameters of the tissue. The measurement of the initial length of the specimen was determined in two ways: it was measured in situ and immediately post dissection, allowing

2 Page 2 of 5 for the effect of rest length measurement on Young s modulus calculation to be examined. The Young s modulus was determined both immediately following elongation and after 20 minutes of stress relaxation. Using the measured in situ length as the rest length, they reported a Young s modulus of 34.6kPa at 40% strain immediately following elongation, dropping to 20.6kPa after 20 minutes of stress relaxation 8. Using post dissection length as the rest length, they reported a Young s modulus of 16.2kPa at 40% strain immediately following elongation, dropping to 8.32kPa after 20 minutes of stress relaxation 8. Perlman et al. 8 indicated that while determining accurate rest length was problematic, measurements made immediately following elongation were most likely the most relevant to phonation studies. Min et al. 9 isolated and examined the Young s modulus of the ligamentous layer of the human vocal fold using two male and two female specimens. They reported, a mean Young s modulus of 600kPa at 40% strain, but their small sample size prevented a gender-based comparison from being explored 9. This study was the first to identify two distinct phases of the vocal fold stress-strain relationship: an initial, low strain, linear segment and a subsequent, high strain, exponential segment. Min et al. 9 used mathematical models to characterize each segment. Chan et al. 10 used histochemistry in addition to longitudinal elongation to compare the effect of collagen and elastin concentration on vocal fold elasticity. Twenty human vocal folds, 12 male and 8 female, were removed from cadaveric specimens, divided into cover and ligamentous layers, and subjected to in vitro sinusoidal deformation. After mechanical testing, the vocal folds were sectioned and examined histologically after tissue staining. Their study showed that male vocal folds contain a higher density of collagen fibres than female, resulting in a higher Young s modulus. Quantitatively, at a 40% strain level, Table 1: Summary of analysed studies utilizing longitudinal elongation to determine Young s modulus. Ishizaka and Kaneko, 1968 Perlman et al., 1984 Min et al., 1995 Chan et al., 2007 Species and Strain (%) the Young s modulus of male vocal folds was determined to be 1000/1750kPa for cover/ligament, respectively, and 480/350kPa for female cover/ligament, respectively 10. They noted that these measurements could indicate that collagen and elastin contribute differentially to vocal fold stiffness. These measurements can be directly compared to the Young s modulus of the vocal fold ligament of 600kPa reported by Min et al. 9 Linear skin rheometry Hess et al. 11 first adapted the technique of linear skin rheometry (LSR), previously used to measure viscoelastic characteristics of the skin, to the measure of the shear modulus of vocal fold tissue (linear skin rheometry results are summarized in table 2). After fixing a piece of vocal fold mucosa to a solid base, a needle probe is suctioned to the tissue, allowing for a sinusoidal, rotational displacement to be applied to the tissue and for shear forces to be extracted. Hess et al. demonstrated that the technique could be used to measure the shear characteristics of the tissue in a repeatable fashion and that the rheometer could distinguish local Values reported for Young's Modulus Human N/A Stiffness constant: 3.7 kpa Canine, 7 40 In situ rest length, imm post elongation: 34.6 kpa 40 In situ rest length, 20 mins post stress relaxation: 20.6 kpa 40 Dissected rest length, imm post elongation: 16.2 kpa 40 Dissected rest length, 20 mins post stress relaxation: 8.32 kpa Human, 4 Low 33.1 kpa kpa kpa Human, Male cover/ligament: 1000/1750kPa 40 Female cover/ligament: 480/350kPa variations in tissue stiffness, as determined by artificially stiffening the tissue at specific locations and examining the shear output. This work was furthered by Goodyer et al. 12 by using LSR to examine the shear modulus of 20 excised, human vocal folds in a hemilarynx model. The hemilarynx model is created by sectioning the larynx in the sagittal plane, which exposes the medial aspect of the vocal fold and allows for easier access for experimentation. By using a hemilarynx, the pre-stress imparted on the tissue by its anatomical surroundings is only partially disrupted, allowing for more accurate estimates of biomechanical properties of the tissue. They reported a mean shear modulus of 1.008kPa and 1.237kPa for male and female larynges, respectively 12. In a more recent study, Goodyer et al. 13 used LSR to examine the stiffness gradient of the subglottal mucosa. Porcine hemilarynges were prepared to expose the subglottis for LSR testing. The measurements were taken at 2mm intervals from the inferior edge of the vocal fold to the superior edge of the trachea. The results of nine hemilarynx samples indicated that the stiffness of the subglottal mucosa approximately doubled as the measurements moved

3 Page 3 of 5 inferiorly to the top of the trachea, a distance with a mean length of 12mm across all specimens. They hypothesized that this stiffness gradient is necessary to ensure an efficient transfer of energy from the airflow produced by the lungs to the mucosal wave. Chhetri et al. 14 used LSR to examine the shear modulus of the cover layer of the vocal fold during intrinsic laryngeal muscle contraction using both an ex vivo human larynx and an in vivo canine larynx. The larynx was kept intact in both specimens, preserving physiologic pre-stress and improving accuracy of shear measurements. For the ex vivo human larynx, muscle contraction was simulated and manipulated using arytenoid adduction sutures and sutures running between the anterior cricoid and the anterior inferior border of the thyroid cartilages to mimic the lateral cricoarytenoid (LCA) and cricothyroid (CT) muscles, respectively. For the in vivo canine larynx, the animal was anesthetized and intubated before the larynx was exteriorized, allowing the rheometer probe to be placed on the vocal fold without oral or pharyngeal hindrance. The bilateral recurrent laryngeal nerves (RLN) and superior laryngeal nerves (SLN) were isolated and fitted with monopolar electrodes, allowing for electrical stimulation of the intrinsic laryngeal muscles during LSR testing. Graded stimulation of the LCA and CT muscles in the human larynx, replicated by increasing suture tension, resulted in increasing the shear modulus to 1.723/4.786kPa, an increase of 1.6/3.7 times the baseline value, respectively 14. They noted that the greater increase in stiffness associated with CT stimulation is in agreement with the body-cover model of the vocal folds [ref Hirano]. With RLN stimulation in the canine larynx, the baseline shear modulus increased to 1.762kPa, an increase of 1.6 times baseline value, and with SLN stimulation, the baseline shear modulus increased to 2.818kPa, an increase of 2.5 times baseline value 14. They noted that while both RLN and SLN stimulation led to increases in the Table 2: Summary of analysed studies utilizing linear skin rheometry to determine shear modulus. Species and shear modulus of the vocal folds, more work was needed to identify the individual laryngeal adductors that generate vocal fold tension. Microindentation Haji et al. 15 first described the microindentation technique as a way to measure the Young s modulus in both human and canine vocal folds at the mid-membranous plane, the anterior commissure, and the vocal process, as well as assessing the elastic characteristics of the ventricular folds (microindentation results are summarized in table 3). In microindentation testing, a solid indenter induces a controlled tissue displacement perpendicular to the surface of the vocal fold tissue, recording resultant forces and allowing for calculation of Young s modulus. By leaving the anatomical relations of the vocal folds unchanged, Haji et al. 15 were able to conclude that the elastic modulus of the midmembranous plane was less than that of the anterior commissure and vocal process, and that the ventricular folds had the lowest stiffness overall. They reasoned that the low stiffness associated with the ventricular folds likely leads to a greater propensity for irregular vibration, leading to production of a rough voice. Tran et al. 16 used a modified microindentation technique as a way to measure the Young s modulus of vocal folds intra-operatively in human patients undergoing laryngeal surgery. The microindenter used in Values reported for Shear Modulus Hess et al., 2006 Human, 1 Dynamic spring rate= g/mm Goodyer et al., 2007 Human, 20 Male: kpa Female: kpa Chhetri et al, 2009 Human, 1 Baseline: kpa ; LCA stimulation: kpa Baseline: kpa; CT stimulation: kpa Canine, 1 Baseline: kpa; RLN stimulation: kpa Baseline: kpa; SLN stimulation: kpa this study consisted of a modified Jako laryngoscope fitted with a depressing plate at one end and a force gauge at the other. By inserting the modified Jako laryngoscope into five anesthetized human patients, stimulating the RLN at either low or high current, and measuring the resultant deflection imparted on the measurement plate, it was possible to extract the local Young s modulus of the tissue rapidly, allowing for translational potential. During experimental testing, Tran et al. 16 collected Young s modulus data for the vocal fold at rest and under high and low RLN stimulation. They reported mean Young s modulus values of 12.6/19.1/21.5kPa for rest/low stimulation/high stimulation respectively. Using the same apparatus Berke and Smith 17,18 measured the Young s modulus of several patients undergoing phonosurgery with vocal fold pathology. Berke and Smith 17,18 studied three patients intra-operatively, two of which received Teflon injections, allowing for a comparison of pre-op and post-op stiffness. They concluded that both Teflon injection pathologic vocal fold fibrosis were associated with increased elastic modulus of the vocal fold. They noted that this modified microindentation technique could provide surgeons with a means to quantitatively assess vocal fold reconstructions and other phonosurgeries throughout the repair process, potentially improving surgical outcome.

4 Page 4 of 5 Chhetri et al. 19 assessed the accuracy of the microindentation technique by creating several different indenters of varying diameters and varying indentation depth, assessing how changes in these variables affected the calculated Young s modulus. They created silicone models of known stiffness, as determined by uniaxial tensile testing, to examine the relationship between indentation depth and indenter diameter, finding that results of microindentation testing were most accurate when the indentation depth was less than or equal to the indenter diameter. Utilizing this paradigm, they tested three excised human vocal fold samples, calculating a mean Young s modulus value of 8.6kPa at the midmembranous plane during low strain testing. Chhetri et al. 19 concluded that the inferior medial surface of the vocal fold was stiffer than the superior medial surface. Chhetri and Rafizadeh 20 recently expanded this work, using microindentation to compare the cover layer stiffness of canine and human vocal folds. Isolating 15 superior medial and 17 inferior medial cover layer samples from eight canine larynges, they reported mean Young s moduli of 4.2kPa and 6.8kPa for the superior medial and inferior medial cover layers, respectively 20. These results were then directly compared to measurements taken from two human cover layers, reporting mean Young s moduli values of 5.0kPa and 7.0kPa for the superior medial and inferior medial cover layers, respectively 20. No statistical difference was calculated between canine and human stiffness at either location. They noted that by demonstrating that the stiffness of canine and human vocal folds was statistically comparable, they could further validate the use of canine larynges as an animal model for laryngeal studies. In a recent study Oren et al. 2 measured the Young s modulus of the superior and inferior aspects of the fold in ex vivo canine larynges using the microindentation technique. Oren et al. 2 kept each larynx intact by Table 3: Summary of analysed studies utilizing microindentation to determine Young s modulus. Species and retracting the fold that was not being tested with a vein retractor, allowing the larynx to retain its physiological pre-stress condition during testing. Their measurements were taken at the mid-membranous plane of 11 canine specimens and showed that the inferior aspect of the vocal fold was consistently stiffer than the superior aspect. They found that the differential stiffness between the inferior and superior aspects of the fold increased at greater strain values, reaching a maximum difference of 35% at 40% strain. They noted that the differential stiffness that exists between the superior and inferior aspect of the vocal fold might contribute to the divergent shape that is formed between the folds during the closing phase of phonation. Discussion The authors have referenced some of their own studies in this review. The protocols of these studies have been approved by the relevant ethics Strain (%) Values reported for Young's Modulus Tran et al., 1993 Human, 5 N/A Rest: 12.6 kpa N/A Low stimulation: 19.1 kpa High stimulation: 21.5 kpa Berke, 1992 Canine, kpa Berke & Smith, 1992 Human, 3 25 Subject 1: 2.38 kpa 50 Subject 1: 5.84 kpa 75 Subject 1: 14.5 kpa 25 Subject 2: 13.3 kpa 50 Subject 2: 19.6 kpa 75 Subject 2: 17.5 kpa 50 Subject 3: 10 kpa Chhetri et al., 2011 Human, 3 Low Intact hemilarynx: 8.6 kpa Low Superior cover: 2.9 kpa Low Medial cover: 4.8 kpa Low Inferior cover: 7.5 kpa Low TA muscle (body): 2.0 kpa Chhetri and Rafizadeh 2013 Canine, 8 N/A Superior medial cover: 4.2 kpa N/A Inferior medial cover: 6.8 kpa Human, 2 N/A Superior medial cover: 5.0 kpa N/A Inferior medial cover: 7.0 kpa Oren et al Canine, Superior aspect: 10 kpa 40 Inferior aspect: 13.6 kpa committees related to the institution in which they were performed. Animal care was in accordance with the institution guidelines. The aforementioned studies detail the current state of research in the field of vocal fold elasticity. While significant progress has been made in the different methodologies of each measurements technique, more work needs to be done to better and more accurately characterize the elastic properties of the vocal fold tissue. The values of the elastic characteristics are a fundamental element that is necessary for laryngeal modelling, which can only be obtained experimentally. Although the work to-date has established the potential of each of these biomechanical methodologies for characterization of vocal fold elasticity, there still remain certain aspects need to be addressed before tissue elasticity becomes useful for clinical diagnostics and therapeutics. Some of these techniques, particularly longitudinal elongation, require excising the vocal fold tissue from the larynx, therefore

5 Page 5 of 5 removing the pre-stress condition that is imparted on the tissue by its anatomical surroundings. This is also a fault of the use of hemilarynges in the characterization of vocal fold stiffness and greatly limits the clinical relevance of the studies that rely on such methodology. Longitudinal elongation is also limited to measurement of the global Young s modulus of the tissue, thus coalescing all tissue anisotropy into a single value for Young s modulus, likely resulting in simplifications of local tissue properties. On the other hand, the LSR and microindentation techniques can resolve the local variations in tissue elasticity, which increase the translational potential of these modalities. Further refinement of these techniques may lead to surgical paradigm shifts and improvements in computational and synthetic laryngeal models. Conclusion Great progress has been made to further understand the inherent tissue properties of the vocal folds, allowing for computational models to more accurately reflect human physiology and surgical corrections to yield better patient outcomes. Further refinement of these measurement techniques may allow for more translational applications of this technology, including the development of new clinical devices designed to characterize the elastic properties of vocal folds in real time. References 1. Titze IR, Talkin DT. A theoretical study of the effects of various laryngeal configurations on the acoustics of phonation. The Journal of the Acoustical Society of America. 1979;66(1): Oren L, Dembinski D, Gutmark E, Khosla S. Characterization of the Vocal Fold Vertical Stiffness in a Canine Model. Journal of Voice Titze IR. Physiologic and acoustic differences between male and female voices. The Journal of the Acoustical Society of America. 1989;85(4): Alipour F, Jaiswal S. Phonatory characteristics of excised pig, sheep, and cow larynges. The Journal of the Acoustical Society of America. 2008;123(6): Alipour F, Jaiswal S, Vigmostad S. Vocal fold elasticity in the pig, sheep, and cow larynges. Journal of Voice. 2011;25(2): Pickup B, Thomson S. Influence of asymmetric stiffness on the structural and aerodynamic response of synthetic vocal fold models. Journal of biomechanics. 2009;42(14): Ishizaka K, Kaneko T. On equivalent mechanical constants of the vocal cords. J Acoust Soc Jpn. 1968;24: Perlman AL, Titze IR, Cooper DS. Elasticity of canine vocal fold tissue. Journal of speech and hearing research. 1984;27(2): Min Y, Titze I, Alipour-Haghighi F. Stress-strain response of the human vocal ligament. The Annals of otology, rhinology, and laryngology. 1995;104 (7): Chan RW, Fu M, Young L, Tirunagari N. Relative contributions of collagen and elastin to elasticity of the vocal fold under tension. Annals of biomedical engineering. 2007;35(8): Hess MM, Mueller F, Kobler JB, Zeitels SM, Goodyer E. Measurements of vocal fold elasticity using the linear skin rheometer. Folia phoniatrica et logopaedica. 2006;58(3): Goodyer E, Hemmerich S, Müller F, Kobler JB, Hess M. The shear modulus of the human vocal fold, preliminary results from 20 larynxes. European archives of oto-rhino-laryngology. 2007;264(1): Goodyer E, Gunderson M, Dailey SH. Gradation of stiffness of the mucosa inferior to the vocal fold. Journal of Voice. 2010;24(3): Chhetri DK, Berke GS, Lotfizadeh A, Goodyer E. Control of vocal fold cover stiffness by laryngeal muscles: a preliminary study. The Laryngoscope. 2009;119(1): Haji T, Mori K, Omori K, Isshiki N. Mechanical properties of the vocal fold. Acta oto-laryngologica. 1992;112 (2): Tran Q, Berke G, Gerratt B, Kreiman J. Measurement of Young's modulus in the in vivo human vocal folds. The Annals of otology, rhinology, and laryngology. 1993;102(8 Pt 1): Berke GS. Intraoperative measurement of the elastic modulus of the vocal fold. Part 1. Device development. The Laryngoscope. 1992; 102(7): Berke GS, Smith ME. Intraoperative measurement of the elastic modulus of the vocal fold. Part 2. Preliminary results. The Laryngoscope. 1992;102 (7): Chhetri DK, Zhang Z, Neubauer J. Measurement of Young's modulus of vocal folds by indentation. Journal of Voice. 2011;25(1): Chhetri DK, Rafizadeh S. Young's Modulus of Canine Vocal Fold Cover Layers. Journal of Voice