Short communicationMicrostructural and mechanical characterization of scarred vocal folds
Introduction
Vocal fold scars can result from the surgical removal of benign or malignant vocal fold lesions, phonotrauma or intubation over an extended period of time (Benninger et al., 1996, Rosen, 2000). Changes in the microstructure and elasticity of the vocal fold lamina propria (LP) hampers the oscillations of the vocal folds. Mechanical injury triggers a cascade of inflammation and healing response, followed by tissue restoration. During wound remodeling, the last phase of the healing process, the extracellular matrix (ECM) of the granulation tissue is reorganized dynamically over months to develop into a mature scar (Hansen and Thibeault, 2006). The concentration, organization, and morphology of collagen, one of the main constituents of the ECM, determine wound stiffness. Published animal studies have shown that collagen is first observed in day 1 (collagen type III) and day 2 (collagen type I) following the surgical removal of the rat LP (Tateya et al., 2006). Total collagen levels peak at weeks 2 and 4, respectively, and then decline to become stable in the period between weeks 8 and 12 (Tateya et al., 2005). Evidence suggests that the remodeling phase in rat vocal fold wounds starts at day 7, undergoes an active remodeling phase between weeks 2 and 4, and reaches a stable remodeling phase at week 8 (Tateya et al., 2005, Tateya et al., 2006).
Changes in collagen morphology alter the tissue elasticity, as manifested by a change in displacement response under tensile loading (Miri et al., 2013). Data reported from animal studies to date include the concentration of structural constituents from immunohistochemistry or molecular data (e.g., see Tateya et al., 2006). The immunohistochemistry methods require time-consuming sample preparation and tissue slicing, which may alter the organization of the collagen fibrils. In the present study, nonlinear laser scanning microscopy (NLSM) based on second harmonic generation (SHG) imaging (Miri et al., 2012b) was used to investigate the morphological changes of collagen in scarred rat vocal folds without physical sectioning of the tissue. Atomic force microscopy (AFM) was also used to quantify the elastic properties of vocal folds through indentation tests. AFM has been utilized for structural characterization at nano and microscales to examine the morphology and elasticity of LP collagen fibrils, as well as the overall elasticity, adhesion, and surface roughness of the LP (Heris et al., 2013, Johanes et al., 2011, Miri et al., 2013). Miri et al. (2013) showed the applicability of AFM and NLSM for better understanding structure-function relationships (and hyperelastic modeling) of normal vocal folds. The present work aimed to use the combination of AFM and NLSM, along with mathematical modeling based on the eight-chain polymer model (Miri et al., 2012a), to characterize vocal fold scarring. This may provide new insights into the role of collagen remodeling in wound healing.
Section snippets
Surgical procedures and sample preparation
The animal study was approved by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison (protocol number MO2358). Vocal fold injuries were created in fourteen Sprague-Dawley adult male rats (4 to 6 months old; 450 to 500 g) following an established protocol (Welham et al., 2009). Briefly, animals were anesthetized and their vocal folds were injured unilaterally using a 25G needle to remove the vocal fold LP. The uninjured side of the vocal fold served as control.
Results
LP removal and associated scarring was first confirmed using standard H&E, as shown in Fig. 1. One month after surgery (Fig. 1a), the scarred vocal fold appeared to have an irregular shape, with a complete epithelium and a fibrous LP. Two months after surgery (Fig. 1b), the gross morphology of the scarred vocal fold approximated that of its uninjured control. Table 1 shows the elastic modulus of uninjured normal controls and injured vocal folds. The statistical differences between elastic
Concluding remarks
The indentation elastic modulus of the uninjured rat LP (250±140 kPa) is much greater than the indentation elastic modulus of porcine LP (3–5 kPa; Heris et al., 2013) and that of the human LP (4–6 kPa; Chhetri et al., 2011). The higher modulus explains why vocalization in rats falls within the ultrasonic-range. The phonatory fundamental frequency of rats (2–4 kHz; Nitschke, 1982) is one order of magnitude greater than that of humans (100–300 Hz; Titze et al., 2003). Contrary to expectations, the
Conflict of interest statement
None.
Acknowledgment
The work has been supported by the National Institute on Deafness and Other Communication Disorders, grant DC 005788 (L. Mongeau, PI), DC 004336 (S. Thibeault, PI), DC012112 (N. Li, PI). The authors would like to express their gratitude to Prof. François Barthelat (Mechanical Engineering Department, McGill University, Montreal) for sharing his atomic force microscope.
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