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Objectives: Endoscopic microlaryngeal laser surgery performed with general anesthesia through a laryngoscope speculum generates heat that accumulates at the distal lumen, creating an "oven" effect and potentially causing bystander thermal damage to nontarget tissue such as the contralateral vocal fold. We report the effects of cooling on air and tissue temperatures that occurred during simulated laryngeal laser surgery with KTP and thulium lasers in an ex vivo calf model. Methods: Ten fresh excised calf larynges were studied at room temperature. Laser energy was applied to one vocal fold for 2 minutes, with or without cooling, while temperatures were monitored with sensors placed within the glottal lumen or inserted superficially into the contralateral vocal fold. A pulsed KTP laser (525 mJ) was used for 5 larynges, and a thulium laser (7 W, continuous) was used for the other 5 larynges. Results: Heating was slightly greater for the KTP laser than for the thulium laser with use of these parameters. The lumen temperatures for both lasers increased an average of 13.2° C without cooling, but only 6.7° C with cooling (p < 0.05). The contralateral vocal fold (subepithelial space) temperature increased an average of 6.8° C without cooling, but only 4.2° C with cooling (p> 0.05). Conclusions: Cooling with room-temperature air during laryngeal laser surgery reduces luminal air and contralateral vocal fold temperatures. This effect is believed to be due to elimination of the plume of steam and smoke that significantly heats surrounding structures. [ABSTRACT FROM AUTHOR]

OBJECTIVES: This study examines the elastic fiber and hyaluronic acid (HA) content of the midmembranous vocal fold laminae propriae (LPs) of humans, dogs, pigs, and ferrets. METHODS: Lamina propria elastin was quantified by measuring the amino acid desmosine, and HA was measured by an enzyme-linked immunosorbent assay-based technique. Quantitative histology was used to evaluate elastin and HA levels in specific LP regions. The distributions of fibrillin-1, a primary microfibrillar component of elastic fibers, and of tropoelastin, an indicator of elastin synthesis, were immunohistochemically analyzed. RESULTS: Elastin and HA constituted 8.5% +/- 2.1% and 0.82% +/- 0.11% of human LP, respectively, relative to tissue total protein. Although the mean LP desmosine levels were similar across species, the mean HA levels in canine (p < 3.1 x 10(-5)), porcine (p < 1.5 x 10(-5)), and ferret (p < 6.6 x 10(-4)) LPs were 3 to 4 times higher than that in humans. Marked interspecies differences in elastin, fibrillin-1, tropoelastin, and HA distributions were observed histologically. CONCLUSIONS: The elastin content of the human LP is roughly twice that of the dermis, whereas the HA content of the human LP is similar to that of the dermis. Although all species had similar levels of desmosine, histologic evaluation indicates that the porcine elastin distribution is most similar to that of the human LP. Fibrillin-1 staining suggests that stress in the human LP may be particularly high in the superior superficial layer, and tropoelastin staining indicates that the rate of LP elastin turnover may vary spatially. [ABSTRACT FROM AUTHOR]

Purpose: Medialization procedures for unilateral vocal fold (VF) paralysis generally improve voice but do not fully replace dynamic VF adduction. Paralyzed VFs typically experience synkinetic reinnervation, which makes it feasible to elicit movement through electrical stimulation. We tested a novel laryngeal pacing implant capable of providing closed-loop (automatic) stimulation of a VF triggered by electromyography (EMG) potentials from the contralateral VF., Method: A custom, battery-powered, microprocessor-based stimulator was tested in eight dogs with bipolar electrodes implanted for recording EMG from the left VF and stimulating adduction of the right VF. A cuff electrode on the left recurrent laryngeal nerve (RLN) stimulated unilateral VF adduction, modeling voluntary control in anesthetized animals. Closed-loop stimulation was tested in both acute and chronic experiments. Synkinetic reinnervation was created in two animals by right RLN transection and suture repair to model unilateral VF paralysis., Results: In all animals, left VF activation through RLN stimulation generated a robust EMG response that rapidly triggered stimulation of contralateral thyroarytenoid and lateral cricoarytenoid muscles, causing nearly simultaneous bilateral adduction. Optimal triggering of VF stimulation from elicited EMG was achieved using independent onset and offset thresholds. Real-time artifact blanking allowed closed-loop stimulation without self-perpetuating feedback, despite the proximity of recording and stimulation electrodes., Conclusions: Using a custom implant system, we demonstrated real-time closed-loop stimulation of one VF triggered by the activation of the contralateral VF. This approach could potentially restore dynamic glottic closure for reflexive behaviors or phonation in cases of unilateral VF paralysis with synkinetic reinnervation., Supplemental Material: https://doi.org/10.23641/asha.24492133.

Objectives: The purpose of this study was to better understand the effects of stitch placement on arytenoid medialization by measuring normative cricoarytenoid joint anatomy and changes in arytenoid position when varying arytenopexy stitch configuration., Methods: This adult human larynx study was done in two parts. First, measurements of the cricoid and arytenoid cartilage anatomy relevant to cricoarytenoid joint function were made in 45 preserved larynges (26 male (M), 19 female (F)) using digital calipers. Second, the arytenoids of six fresh larynges ( three M, three F) were sutured to the cricoid using various arytenopexy-stitch placements ranging from inferior-lateral to superior-medial, and the resulting arytenoid positions were compared by measuring medial displacement of the arytenoid body and change in glottal configuration from macro still images using Image J. Paired t-tests were used to compare the results., Results: Cartilage and joint facet dimensions showed differences between males (M) and females (F). Cricoid facet lengths averaged 9.3 mm (M) and 7.1 mm (F), and widths averaged 4.9 mm (M) and 4.0 mm (F). The arytenoid facet widths averaged 10.5 mm (M) and 9.7 mm (F). Average distances between cricoid facets were 11.8 mm for both males and females. Securing the arytenoid superior-medially on the cricoid facet produced more medialization (2.2 mm vs 1.0 mm, P < .001) and better glottic aperture configuration (9.5° vs 2.7°, P < .001) than securing the arytenoid inferior-laterally on the facet., Conclusions: Anatomic consistency in cricoarytenoid anatomy provides reliable surgical landmarks for ideal placement of an arytenopexy suture to optimally reposition the arytenoid cartilage. Optimal arytenoid medialization can be accurately reproduced with an arytenopexy-suture that is placed superior-medially on the cricoid facet., Level of Evidence: NA Laryngoscope, 131:2540-2544, 2021., (© 2021 The American Laryngological, Rhinological and Otological Society, Inc.)

The purpose of this paper is to report on the first in vivo application of a recently developed transoral, dual-sensor pressure probe that directly measures intraglottal, subglottal, and vocal fold collision pressures during phonation. Synchronous measurement of intraglottal and subglottal pressures was accomplished using two miniature pressure sensors mounted on the end of the probe and inserted transorally in a 78-year-old male who had previously undergone surgical removal of his right vocal fold for treatment of laryngeal cancer. The endoscopist used one hand to position the custom probe against the surgically medialized scar band that replaced the right vocal fold and used the other hand to position a transoral endoscope to record laryngeal high-speed videoendoscopy of the vibrating left vocal fold contacting the pressure probe. Visualization of the larynx during sustained phonation allowed the endoscopist to place the dual-sensor pressure probe such that the proximal sensor was positioned intraglottally and the distal sensor subglottally. The proximal pressure sensor was verified to be in the strike zone of vocal fold collision during phonation when the intraglottal pressure signal exhibited three characteristics: an impulsive peak at the start of the closed phase, rounded peak during the open phase, and minimum value around zero immediately preceding the impulsive peak of the subsequent phonatory cycle. Numerical voice production modeling was applied to validate model-based predictions of vocal fold collision pressure using kinematic vocal fold measures. The results successfully demonstrated feasibility of in vivo measurement of vocal fold collision pressure in an individual with a hemilaryngectomy, motivating ongoing data collection that is designed to aid in the development of vocal dose measures that incorporate vocal fold impact collision and stresses., Competing Interests: Conflicts of Interest: Drs. Robert Hillman, Steven Zeitels, and Daryush Mehta have a financial interest in InnoVoyce LLC, a company focused on developing and commercializing technologies for the prevention, diagnosis and treatment of voice-related disorders. Dr. Hillman’s, Dr. Zeitels’, and Dr. Mehta’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. Dr. Matías Zañartu has a financial interest in Lanek SPA, a company focused on developing and commercializing biomedical devices and technologies. Dr. Zañartu’s interests were reviewed and are managed by Universidad Técnica Federico Santa María in accordance with its conflict-of-interest policies. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Objective: Tracking recovery after nerve injury may require many intermittent assessments over long periods, preferably with non- or minimally invasive methods. We developed subcutaneous electrical connection ports (ECPs) for repeated connection to nerve cuff or intramuscular electrodes via transdermal needles and evaluated them during studies of laryngeal reinnervation., Study Design: Animal experiment., Setting: Laboratory., Methods: ECPs were designed and 3-dimensionally printed for connection to bipolar electrodes with biocompatible polymers. Dual compartments filled with conductive silicone capped with nonconductive silicone were used to make the connections between electrode leads and transdermally inserted needles. Ten dogs (19-29 kg) were implanted with 22 ECPs. In 7 dogs, 11 electrodes were placed on recurrent laryngeal nerves proximal to transection and suture repair to track laryngeal reinnervation. In 6 dogs, 8 spinal accessory nerve cuff electrodes were used to stimulate neck muscle contraction. In 2 dogs, 3 electrodes were implanted in the thyroarytenoid muscle. Stimulation thresholds, electromyography, and videolaryngoscopic imaging were obtained in 156 tests over survival periods up to 32 months. Stimulation data provided information about ECP performance., Results: ECPs added negligible resistance to electrodes (mean ± SD, 2.14 ± 0.9 Ω). Despite some electrode leads breaking distally, ECPs were reliable and well tolerated at implant sites and enabled periodic assessment of nerve and muscle function over the time course of laryngeal reinnervation. Histology showed ECP encapsulation as thin layers of connective tissue and minimal acute inflammation., Conclusion: Custom ECPs are easily fabricated and cause little tissue reaction over months to years of subcutaneous implantation, facilitating long-term physiologic studies.

The ability to provide absolute calibrated measurement of the laryngeal structures during phonation is of paramount importance to voice science and clinical practice. Calibrated three-dimensional measurement could provide essential information for modeling purposes, for studying the developmental aspects of vocal fold vibration, for refining functional voice assessment and treatment outcomes evaluation, and for more accurate staging and grading of laryngeal disease. Recently, a laser-calibrated transnasal fiberoptic endoscope compatible with high-speed videoendoscopy (HSV) and capable of providing three-dimensional measurements was developed. The optical principle employed is to project a grid of 7 × 7 green laser points across the field of view (FOV) at an angle relative to the imaging axis, such that (after calibration) the position of each laser point within the FOV encodes the vertical distance from the tip of the endoscope to the laryngeal tissues. The purpose of this study was to develop a precise method for vertical calibration of the endoscope. Investigating the position of the laser points showed that, besides the vertical distance, they also depend on the parameters of the lens coupler, including the FOV position within the image frame and the rotation angle of the endoscope. The presented automatic calibration method was developed to compensate for the effect of these parameters. Statistical image processing and pattern recognition were used to detect the FOV, the center of FOV, and the fiducial marker. This step normalizes the HSV frames to a standard coordinate system and removes the dependence of the laser-point positions on the parameters of the lens coupler. Then, using a statistical learning technique, a calibration protocol was developed to model the trajectories of all laser points as the working distance was varied. Finally, a set of experiments was conducted to measure the accuracy and reliability of every step of the procedure. The system was able to measure absolute vertical distance with mean percent error in the range of 1.7% to 4.7%, depending on the working distance., (Copyright © 2019 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives/hypothesis: We previously developed an instrument called the Aerodynamic Vocal Fold Driver (AVFD) for intraoperative magnified assessment of vocal fold (VF) vibration during microlaryngoscopy under general anesthesia. Excised larynx testing showed that the AVFD could provide useful information about the vibratory characteristics of each VF independently. The present investigation expands those findings by testing new iterations of the AVFD during microlaryngoscopy in the canine model., Study Design: Animal model., Methods: The AVFD is a handheld instrument that is positioned to contact the phonatory mucosa of either VF during microlaryngoscopy. Airflow delivered through the AVFD shaft to the subglottis drives the VF into phonation-like vibration, which enables magnified observation of mucosal-wave function with stroboscopy or high-speed video. AVFD-driven phonation was tested intraoperatively (n = 26 VFs) using either the original instrument design or smaller and larger versions three-dimensionally printed from a medical grade polymer. A high-fidelity pressure sensor embedded within the AVFD measured VF contact pressure. Characteristics of individual VF phonation were compared with typical two-fold phonation and compared for VFs scarred by electrocautery (n = 4) versus controls (n = 22)., Results: Phonation was successful in all 26 VFs, even when scar prevented conventional bilateral phonation. The 15-mm-wide AVFD fits best within the anteroposterior dimension of the musculo-membranous VF, and VF contact pressure correlated with acoustic output, driving pressures, and visible modes of vibration., Conclusions: The AVFD can reveal magnified vibratory characteristics of individual VFs during microlaryngoscopy (e.g., without needing patient participation), potentially providing information that is not apparent or available during conventional awake phonation, which might facilitate phonosurgical decision making., Level of Evidence: NA Laryngoscope, 130: 1980-1988, 2020., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

A critical element in understanding voice production mechanisms is the characterization of vocal fold collision, which is widely considered a primary etiological factor in the development of common phonotraumatic lesions such as nodules and polyps. This paper describes the development of a transoral, dual-sensor intraglottal/subglottal pressure probe for the simultaneous measurement of vocal fold collision and subglottal pressures during phonation using two miniature sensors positioned 7.6 mm apart at the distal end of a rigid cannula. Proof-of-concept testing was performed using excised whole-mount and hemilarynx human tissue aerodynamically driven into self-sustained oscillation, with systematic variation of the superior-inferior positioning of the vocal fold collision sensor. In the hemilarynx experiment, signals from the pressure sensors were synchronized with an acoustic microphone, a tracheal-surface accelerometer, and two high-speed video cameras recording at 4000 frames per second for top-down and en face imaging of the superior and medial vocal fold surfaces, respectively. As expected, the intraglottal pressure signal exhibited an impulse-like peak when vocal fold contact occurred, followed by a broader peak associated with intraglottal pressure build-up during the de-contacting phase. As subglottal pressure was increased, the peak amplitude of the collision pressure increased and typically reached a value below that of the average subglottal pressure. Results provide important baseline vocal fold collision pressure data with which computational models of voice production can be developed and in vivo measurements can be referenced., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Objectives/hypothesis: Photoangiolytic precision afforded by the 532-nm potassium-titanyl-phosphate (KTP) laser relies on predictable energy delivery. Inadequate energy output can cause vessel rupture, and excessive energy can cause thermal damage. The quality of the cleaved surface and carbon deposits from ablated tissue are two factors that could negatively impact fiber performance. The effects of these on energy output and blood vessel coagulation were assessed using a chorioallantoic membrane (CAM) model., Study Design: Comparative analysis., Methods: Laser fibers with carbon debris, optimal fiber cleaving, and suboptimal cleaving were inspected at three times magnification, and the light dispersion pattern of each fiber was rated. The average energy output from consecutive pulses through each fiber configuration was recorded. The effect of these fiber conditions on clinical efficacy was estimated by measuring vessel coagulation versus rupture in the CAM model. Repeated measures analysis of variance compared results., Results: Carbon debris and suboptimal cleaving resulted in decreased energy output in comparison to optimal cleaving ([-Δ244 mJ, d = 4.31, P < .001] and [-Δ195 mJ, d = 6.04, P < .001]). Optimal cleaving resulted in immediate coagulation of vessels. Fibers with suboptimal cleaving and carbon debris had unpredictable outcomes, requiring multiple pulses for coagulation or causing vessel rupture., Conclusions: KTP laser fiber function is significantly affected by fiber tip condition. Carbon debris and suboptimal cleaving create significant attenuation of energy, which results in an unpredictable angiolytic effect, as demonstrated by increased vessel rupture in the CAM model. Optimal recleaving of KTP laser fibers restores prior energy output and predictable coagulation. Care should be taken to avoid carbon debris on laser-fiber tips and to cleave fibers properly., Level of Evidence: NA Laryngoscope, 129:2244-2248, 2019., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

Excessive vocal fold collision pressures during phonation are considered to play a primary role in the formation of benign vocal fold lesions, such as nodules. The ability to accurately and reliably acquire intraglottal pressure has the potential to provide unique insights into the pathophysiology of phonotrauma. Difficulties arise, however, in directly measuring vocal fold contact pressures due to physical intrusion from the sensor that may disrupt the contact mechanics, as well as difficulty in determining probe/sensor position relative to the contact location. These issues are quantified and addressed through the implementation of a novel approach for identifying the timing and location of vocal fold contact, and measuring intraglottal and vocal fold contact pressures via a pressure probe embedded in the wall of a hemi-laryngeal flow facility. The accuracy and sensitivity of the pressure measurements are validated against ground truth values. Application to in vivo approaches are assessed by acquiring intraglottal and VF contact pressures using a synthetic, self-oscillating vocal fold model in a hemi-laryngeal configuration, where the sensitivity of the measured intraglottal and vocal fold contact pressure relative to the sensor position is explored., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Objectives/hypothesis: Lubricin/proteoglycan-4 (PRG4) lubricates connective tissues such as joints and tendon sheaths, enabling them to better withstand shearing and frictional forces during motion. We wondered whether PRG4 might play a role in phonation, as normal vocal folds withstand repetitive, high-velocity deformations remarkably well. As a first step, we tested whether PRG4 is expressed in vocal folds., Study Design: Laboratory study., Methods: Anatomical and molecular methods were applied to 47 larynges from humans, macaque (Macaca fascicularis), canines, pigs, calves, and rats. Immunohistochemistry (IHC), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) methods were used to test for the presence of PRG4., Results: In all species, the true vocal fold lamina propria (TVF-LP) was positive for PRG4 by IHC, whereas immunoreactivity of the false vocal fold was weak or absent, depending on the species. Human TVF-LP was strongly stained across all layers. Immunoreactivity was seen variably on the vocal fold surface and within the vocal fold epithelium, in the conus elasticus and thyroglottic ligament, and at the tip of vocal process. Western blots of four humans and six pigs demonstrated immunoreactivity at appropriate molecular weight. qRT-PCR of pig tissues confirmed PRG4 mRNA expression, which was highest in the TVF-LP., Conclusions: PRG4 was found in phonatory tissues of six mammals. We suggest it might act as a lubricant within the lamina propria and possibly on the vocal fold surface, limiting phonation-related damage to vocal fold extracellular matrix and epithelium, and enhancing vocal efficiency by reducing internal friction (viscosity) within the vocal fold., Level of Evidence: NA Laryngoscope, 129:E229-E237, 2019., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

Objective:: The goal of this work was to create implantable stimulator systems that could be used in animal experiments on laryngeal paralysis, including "closed-loop" stimulation of impaired muscles triggered by electromyographic (EMG) potentials from healthy muscles., Study Design:: Iterative device design and testing., Methods:: A series of microcontroller-based implantable devices were built that incorporated increasingly sophisticated features for stimulation, EMG recording, and communication across the skin. Specific engineering challenges included minimizing power consumption, achieving charge-balanced and relatively high stimulation capacity, implementing noninvasive communication across the skin, providing real-time processing of EMG signals, and mitigating effects of shock artifacts. Bench testing was used to verify performance., Results:: Two prototypes are described in detail. Each system is based on an "implant" and an external "communication adapter" that interfaces both with the implant and with external computers for adjustments and monitoring. The first version described is inductively powered and referred to as the "inductive laryngeal stimulator." It uses inductive coupling for both power and communication and performs EMG processing in the communication adapter module. The second version, a "battery-powered laryngeal stimulator," consists of an autonomous battery-powered implant with onboard EMG processing and artifact control; it communicates by infrared light with the external communication adapter for setup and monitoring., Conclusions:: The devices met design and performance specifications and have proved useful in the animal experiments that are described in Part 2 of this series. Detailed descriptions of the circuits and their firmware are made available in the Appendix. Level of Evidence: NA.

Objective:: Laryngeal paralysis of central or peripheral origin can potentially be treated using functional electrical stimulation (FES) of laryngeal muscles. Experiments in canines (dogs) were performed using implant prototypes capable of closed-loop FES to refine engineering designs and specifications, test surgical approaches for implantation, and better understand the in vivo effects of laryngeal muscle stimulation on short- and long-term glottic function., Study Design:: Prospective, laboratory., Methods:: We designed and tested a series of microprocessor-based implantable devices that can stimulate glottic opening or closing based on input from physiological control signals (real-time processing of electromyographic [EMG] signals). After acute device testing experiments, 2 dogs were implanted for 8 and 24 months, with periodic testing of closed-loop laryngeal muscle stimulation triggered from EMG signals. In total, 5 dogs were tested for the effects of laryngeal muscle stimulation on vocal fold (VF) posturing in larynges with nerve supplies that were intact (7 VFs), synkinetically reinnervated (2 VFs), or chronically denervated (1 VF). In 3 cases, the stimulation was combined with airflow-driven phonation to study the consequent modulation of phonatory parameters., Results:: Initial device prototypes used inductive coupling for power and communication, while later iterations used battery power and infrared light communication (detailed descriptions are provided in the Part 1 companion paper). Two animals were successfully implanted with the inductively powered units, which operated until removed at 8 months in 1 animal or for more than 16 months in the second animal. Surgically, the encapsulated implants were well tolerated, and procedures for placing, attaching, and connecting the devices were developed. To simulate EMG control signals in anesthetized animals, we created 2 types of nerve/muscle signal sources. In one approach, a neck muscle had a cuff electrode placed on its motor nerve that was connected to transdermal electrical connection ports for periodic testing. In the second approach, the recurrent laryngeal nerve on one side of the larynx was stimulated to generate a VF EMG signal, which was then used to trigger FES of the paralyzed contralateral side (eg, restoring VF movement symmetry). Implant testing identified effective stimulation parameters and closed-loop stimulation artifact rejection techniques for FES of both healthy and paralyzed VFs. Stimulation levels effective for VF adduction did not cause signs of discomfort during awake testing., Conclusion:: Our inductive and battery-powered prototypes performed effectively during in vivo testing, and the 2 units that were implanted for long-term evaluation held up well. As a proof of concept, we demonstrated that elicited neck strap muscle or laryngeal EMG potentials could be used as a control signal for closed-loop stimulation of laryngeal adduction and vocal pitch modulation, depending on electrode positioning, and that VFs were stimulable in the presence of synkinetic reinnervation or chronic denervation.

Objectives:: Gels composed of carboxymethylcellulose (CMC) and cross-linked hyaluronic acid are commonly used as temporary or resorbable injectable materials for vocal fold medialization. However, there is limited information about tissue injection patterns, soft tissue reaction, degradation, and residence time, particularly for the newer CMC gels., Study Design:: Prospective, laboratory., Methods:: Ten canines underwent paraglottic (deep to the vocal ligament) microlaryngoscopic injection with about 100 µL (0.1 mL) of cross-linked hyaluronic acid (Restylane) in the right vocal fold and about 100 µl (0.1 mL) of CMC gel in the left vocal fold. Two commercial CMC gels of similar formulation (Prolaryn Gel and Renú Gel) were used in 5 animals each. Two subjects were sacrificed for histologic analysis at 3, 13, 29, 42, and 55 days., Results:: Histology showed that injected Restylane formed cohesive ovoid-shaped aggregated implants with minimal dispersion, inflammation, or cellular invasion in all subjects. Approximately 86% of Restylane injected remained at 8 weeks, as estimated from postmortem, high-resolution magnetic resonance imaging scans. In contrast, histology showed that both CMC gels dispersed widely through fascial planes during and after injection within and outside the thyroarytenoid muscle fascicles. There was a robust macrophage and histiocyte phagocytic response, with moderate to severe inflammation noted around residual CMC gel at early time points, and at 6 to 8 weeks, very little free gel was observed., Conclusions:: None of the studied materials induced a clinical inflammatory reaction on laryngoscopy such that they would be considered problematic or dangerous. Restylane demonstrated superior injection localization, tissue compatibility, and residence time. Both CMC gels did not localize well in the paraglottic region, and there was a robust inflammatory response, with clearance by macrophages and short residence time. These results suggest that CMC gels may have a more limited application than previously thought for vocal fold medialization in patients in whom the injection is being used to achieve voice enhancement while awaiting reinnervation. Level of Evidence: NA.

Introduction:: Endotracheal (ET) intubation is a common cause of acquired glottic stenosis. Severe cases often require an irreversible arytenoidectomy/cordectomy, which typically results in poor voice quality. Adult human cadaver larynges were studied to gain insights about ET tube-induced posterior glottic injuries, hoping to create a less invasive remedy., Study Design:: Human cadaver investigation and case reports., Methods:: Microlaryngeal assessments were done on 10 human cadaver larynges (5 men, 5 women) with and without ET tubes. After supracricoid soft tissue resection, measurements were obtained, including the distance between the outer diameter of the ET tube and the medial aspect of the cricoarytenoid joint facet. Additionally, measurements of the circumferential arc of differently sized ET tubes were made alongside both cricoarytenoid joint capsules. This information was used to design a silastic stent that would function as a self-retaining interarytenoid spring to treat posterior glottic stenosis in 5 patients. Four of 5 patients included in the clinical study were tracheotomy dependent, primarily because of glottic stenosis. The human surgical technique is described in detail., Results:: The shortest distance between the outer diameter of the ET tube to the medial cricoid facet averaged 5.02 mm in men and 3.62 mm in women. On the basis of the diameter of the intralaryngeal component of the initially round stent, and the position of the cricoarytenoid joint facets, the interarytenoid spring would have a subtended arc between 110° and 175°. These data helped fashion parameters for modifying a conventional T-tube to form a new self-retaining silastic interarytenoid spring. The first 5 human cases have been successful, allowing effective tracheotomy tube decannulation and excellent voice quality., Conclusions:: The anatomic investigation herein provided key insights into ET tube-induced glottic stenosis and facilitated a new straightforward procedure to surgically improve the airway yet preserve excellent vocal function in patients with acquired glottic stenosis. Level of Evidence: NA.

Objectives: To compare the physical characteristics of 3 laser-protected endotracheal tubes (LPETs) commonly used in endoscopic laser surgery. To report potential intraoperative problems related to LPET use and suggest practical solutions., Study Design: Comparative analysis., Setting: Academic laboratory., Subjects and Methods: Physical characteristics of the Mallinckrodt Laser-Flex (MTL), Medtronic Laser-Shield II (ML-II), and Rusch LaserTubus (RL) were compared. The effect of bending LPETs on airflow resistance was estimated with a pressure transducer. The force required to pull each tube through the glottis and the pressure exerted during this maneuver were measured in a fresh cadaveric human larynx., Results: The design features and physical characteristics of LPETs differ, including varying balloon-tip lengths. Bending LPETs to acute angles caused significant pressure increase within the RL tube (Δ 3.42 cm H 2 O) and minimal change within the ML-II (Δ 0.12 cm H 2 O) and MTL (Δ 0.21 cm H 2 O) tubes. The average force required to pull the RL (48.12 g, P = .003) and MTL (282.4 g, P = .001) tubes through the glottis was 7.6× and 44.5× greater than that for the ML-II (6.39 g). When pulled through the vocal folds, the ML-II cuff exerted no detectable pressure, whereas higher pressures were measured for the RL (2.2 cm H 2 O) and MTL (6.5 cm H 2 O) tubes., Conclusion: The ML-II tube had the most favorable characteristics, with minimal pressure during extubation and resistance to kinking. The RL tube kinks readily with a resultant increase in resistance to airflow. The MTL tube extends farther into the trachea due to a relatively elongated balloon-tip configuration. Future LPET designs should incorporate features that avoid intraoperative difficulties related to airway protection and ventilation.

Objectives: Aortic homografts integrate well with laryngeal tissue when used in reconstructive surgery. It was hypothesized that a paste of aortic homograft, rich in slow-to-degrade elastin, would compare favorably in residence time and biocompatibility to predicate materials used for vocal fold injection-medialization., Methods: An injectable aorta paste (AP) was made by pulverizing aortic homografts at -196°C (cryomilling). To assess residence time and biocompatibility, 0.3 cc was injected subdermally in guinea pigs (n = 3 per 2-, 4-, 8-, 16-, 24-week time points) followed by histological analysis. To test particle size versus residence time, APs made using 80 or 200 seconds of cryomilling were compared. Implant characteristics of AP were then compared to Restylane, Radiesse Voice (Hydroxylapatite), Radiesse Voice Gel, and Cymetra in additional animals (n = 6 per 4-, 8-, 12-week time points)., Results: Injected AP formed ovoid masses with minimal inflammation. Cellular infiltration was mild and increased with survival time. There was a gradual reduction of implant volume to ~40% at 24 weeks. Increased residence time for paste with larger particles (80 cryomilling seconds) was noted. Von Kossa staining showed progressive calcification of the AP. Cymetra was difficult to reconstitute reliably but formed subdermal masses similar to AP in shape, size, and reactivity and without calcification. The other predicates showed good biocompatibility but spread more widely and erratically in the tissue., Conclusion: Aortic paste is easy to create, biocompatible, degrades slowly, and forms well-defined implants in guinea pig subdermal tissue. The AP implants calcified over time, and experiments are ongoing to determine the source of calcification and how it might be controlled or exploited clinically., (© The Author(s) 2016.)

The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p < 0.05) than control tissue (0.73 ± 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p < 0.005) fiber density (0.72 ± 0.09) compared to controls (0.18 ± 0.03). In the present study, image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as application of steroids or drugs targeting pathways involved in fibrosis. SCANNING 38:684-693, 2016. © 2016 Wiley Periodicals, Inc., (© Wiley Periodicals, Inc.)

Vocal fold kinematics and its interaction with aerodynamic characteristics play a primary role in acoustic sound production of the human voice. Investigating the temporal details of these kinematics using high-speed videoendoscopic imaging techniques has proven challenging in part due to the limitations of quantifying complex vocal fold vibratory behavior using only two spatial dimensions. Thus, we propose an optical method of reconstructing the superior vocal fold surface in three spatial dimensions using a high-speed video camera and laser projection system. Using stereo-triangulation principles, we extend the camera-laser projector method and present an efficient image processing workflow to generate the three-dimensional vocal fold surfaces during phonation captured at 4000 frames per second. Initial results are provided for airflow-driven vibration of an ex vivo vocal fold model in which at least 75% of visible laser points contributed to the reconstructed surface. The method captures the vertical motion of the vocal folds at a high accuracy to allow for the computation of three-dimensional mucosal wave features such as vibratory amplitude, velocity, and asymmetry.

Objective: This study aimed to develop and evaluate a model for teaching transcervical laryngeal injections., Methods: A 3-dimensional printer was used to create a laryngotracheal framework based on de-identified computed tomography images of a human larynx. The arytenoid cartilages and intrinsic laryngeal musculature were created in silicone from clay casts and thermoplastic molds. The thyroarytenoid (TA) muscle was created with electrically conductive silicone using metallic filaments embedded in silicone. Wires connected TA muscles to an electrical circuit incorporating a cell phone and speaker. A needle electrode completed the circuit when inserted in the TA during simulated injection, providing real-time feedback of successful needle placement by producing an audible sound. Face validation by the senior author confirmed appropriate tactile feedback and anatomical realism. Otolaryngologists pilot tested the model and completed presimulation and postsimulation questionnaires., Results: The high-fidelity simulation model provided tactile and audio feedback during needle placement, simulating transcervical vocal fold injections. Otolaryngology residents demonstrated higher comfort levels with transcervical thyroarytenoid injection on postsimulation questionnaires., Conclusion: This is the first study to describe a simulator for developing transcervical vocal fold injection skills. The model provides real-time tactile and auditory feedback that aids in skill acquisition. Otolaryngologists reported increased confidence with transcervical injection after using the simulator., (© The Author(s) 2014.)

Background: Obesity is a major risk factor for the development of diabetes. Limb ischemia-reperfusion injury (IR) is a common clinical problem in diabetics who have compromised lower extremity perfusion. This study compared the histologic, metabolic, and functional outcomes after hind limb IR in diet-induced obese (DIO) and non-diabetic (ND) mice during the acute and the regenerative phases of IR., Methods: DIO and ND mice were subjected to 1.5 h unilateral hind limb ischemia followed by 1- or 28-d IR. Muscle morphology, metabolic, and genomic stress were evaluated at days 1 and 28 IR; Acute inflammation and thrombosis were only measured at day-1 IR. At day 28, IR, skeletal muscle contractility, and maturation were also assessed., Results: At day-1 IR, similar levels of acute muscle fiber necrosis were seen in both groups. DIO mice demonstrated substantially greater inflammatory, prothrombotic, and genomic stress responses, which were also associated with a greater reduction in energy substrates and Akt phosphorylation. At 28d, there was no difference in the peak forces generated in the hind limbs for the two groups. DIO mice had reduced fatigue resistance compared with ND and larger areas of fat accumulation although there was no significant difference in muscle fiber maturation., Conclusions: DIO mice had an exacerbated acute response to IR with enhanced metabolic deficit, fat accumulation, and defective functional recovery during the regenerative phase of IR. These changes in fatigue resistance reflect compromised functional recovery after IR injury and have relevance for the functional recovery of patients with metabolic syndrome and insulin resistance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Particle size, stiffness and surface functionality are important in determining the injection site, safety and efficacy of injectable soft-tissue fillers. Methods to produce soft injectable biomaterials with controlled particle characteristics are therefore desirable. Here we report a method based on suspension photopolymerization and semi-interpenetrating network (semi-IPN) to synthesize soft, functionalizable, spherical hydrogel microparticles (MP) of independently tunable size and stiffness. MP were prepared using acrylated forms of polyethylene glycol (PEG), gelatin and hyaluronic acid. Semi-IPN MP of PEG-diacrylate and PEG were used to study the effect of process parameters on particle characteristics. The process parameters were systematically varied to produce MP with size ranging from 115 to 515μm and stiffness ranging from 190 to 1600Pa. In vitro studies showed that the MP thus prepared were cytocompatible. The ratio and identity of the polymers used to make the semi-IPN MP were varied to control their stiffness and to introduce amine groups for potential functionalization. Slow-release polymeric particles loaded with Rhodamine or dexamethasone were incorporated in the MP as a proof-of-principle of drug incorporation and release from the MP. This work has implications in preparing injectable biomaterials of natural or synthetic polymers for applications as soft-tissue fillers., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objectives: Mandibular tori have been identified as a contributing factor in difficult exposure during intubation. However, no investigation has measured the effect of mandibular tori on glottic exposure during suspension microlaryngoscopy (SML). The objective of this study was to measure how the size and location of mandibular tori affect glottic exposure during simulated SML at different thyromental distances., Methods: Suspension microlaryngoscopy was modeled on an anatomically accurate skull and larynx with thyromental distances between 6 and 12 cm. Mandibular tori were simulated by protruding screws 5 to 15 mm from the lingual aspect of the mandible. The tori were positioned either 15 mm (anterior) or 25 mm (posterior) from the midline of the symphysis. The glottic exposure for the various-size tori in each location was measured by recording the displacement of the glottiscope tip relative to the most anterior exposure achievable without tori. The glottiscope angle relative to the horizontal plane was measured for each condition., Results: Mandibular tori of more than 10 mm had a significant impact on glottic exposure. Displacement of the glottiscope tip ranged from 2 to 9 mm for anteriorly placed tori and from 7 to 29 mm for posteriorly placed tori, with larger tori causing greater displacement. Increasing the thyromental distance increased the posterior glottiscope tip displacement regardless of torus size or location. The glottiscope angle increased with larger tori (12º to 28º), but this angle did not change with increasing thyromental distance., Conclusions: Larger size and more-posterior location of mandibular tori more significantly reduce glottic exposure during SML. The inner table of the mandible is the most relevant anatomic constraint on glottic exposure, which varies with the presence or absence of mandibular tori independent of thyromental distance.

Functional recovery is typically poor after facial nerve transection and surgical repair. In rats, whisking amplitude remains greatly diminished after facial nerve regeneration, but can recover more completely if the whiskers are periodically mechanically stimulated during recovery. Here we present a robotic "whisk assist" system for mechanically driving whisker movement after facial nerve injury. Movement patterns were either preprogrammed to reflect natural amplitudes and frequencies, or movements of the contralateral (healthy) side of the face were detected and used to control real-time mirror-like motion on the denervated side. In a pilot study, 20 rats were divided into nine groups and administered one of eight different whisk assist driving patterns (or control) for 5-20 minutes, five days per week, across eight weeks of recovery after unilateral facial nerve cut and suture repair. All rats tolerated the mechanical stimulation well. Seven of the eight treatment groups recovered average whisking amplitudes that exceeded controls, although small group sizes precluded statistical confirmation of group differences. The potential to substantially improve facial nerve recovery through mechanical stimulation has important clinical implications, and we have developed a system to control the pattern and dose of stimulation in the rat facial nerve model.

Efficient transfer of sound by the middle ear ossicles is essential for hearing. Various pathologies can impede the transmission of sound and thereby cause conductive hearing loss. Differential diagnosis of ossicular disorders can be challenging since the ossicles are normally hidden behind the tympanic membrane (TM). Here we describe the use of a technique termed optical coherence tomography (OCT) vibrography to view the sound-induced motion of the TM and ossicles simultaneously. With this method, we were able to capture three-dimensional motion of the intact TM and ossicles of the chinchilla ear with nanometer-scale sensitivity at sound frequencies from 0.5 to 5 kHz. The vibration patterns of the TM were complex and highly frequency dependent with mean amplitudes of 70-120 nm at 100 dB sound pressure level. The TM motion was only marginally sensitive to stapes fixation and incus-stapes joint interruption; however, when additional information derived from the simultaneous measurement of ossicular motion was added, it was possible to clearly distinguish these different simulated pathologies. The technique may be applicable to clinical diagnosis in Otology and to basic research in audition and acoustics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: We assessed the likelihood of arytenoid dislocation during intubation through the application of controlled force., Methods: Six cadaveric human larynges were mounted in an apparatus for simulating forcible collision with the arytenoid complexes. An endotracheal tube tip probe (ETTP) was used to push one arytenoid complex, and a non-slip probe (NSP) was tested on the other. Increasing pressure was applied until the probes either slipped or reached 5 kg of force. Dissection was then performed to assess the integrity of the cricoarytenoid ligament. The forces obtained by pushing an endotracheal tube against an electronic balance were measured to estimate the maximal possible intubating force., Results: None of the ETTP or NSP trials disrupted the cricoarytenoid joint ligaments, and the joint never appeared to be dislocated. The mean maximal forces were 1.8 kg for the ETTP (after which, slippage consistently occurred) and 4.7 kg for the NSP. The mean maximal forces from an endotracheal tube pushed against a scale were 1.5 kg (without stylet) and 4.6 kg (with stylet)., Conclusions: Arytenoid dislocation did not happen, and gross disruption of the joint capsule or ligament did not occur, even when the testing approximated the maximum force achievable under extreme conditions. Endotracheal tube insertion thus seems unlikely to cause arytenoid dislocation.

Objectives/hypothesis: Vocal fold (VF) injections of viscous materials are typically performed using hand-operated syringes or injection guns; however, these methods can be imprecise due to accumulation of pressure, effort-related tremor, and poor feedback regarding injection volume and rate., Study Design: Apparatus development with laboratory bench-top and animal model testing., Methods: A foot pedal-triggered device for dispensing viscous materials was modified by adding a linear transducer and display for monitoring dispensed volume. In bench tests, bovine VFs were injected with fluids/materials of different viscosities (saline, glycerol, hydrogel, and liposuctioned fat) through narrow-bore needles using a range of driving pressures and air pulse durations. The device was further evaluated in >50 in vivo VF injection experiments., Results: Device function was repeatable, with high correlations (typically R(2) > 0.98) between the readout and direct measures of volume, even for small volumes (<5 μL/pulse). Foot pedal control enabled surgeons to make steady, accurate injections into ferret and dog VFs during phonosurgery, and, because the dispenser released all driving pressure between pulses, there were no instances of clog-related overinjection when the obstruction cleared., Conclusions: This VF injection system shows promise for development to enhance human phonosurgery by increasing injection control and precision., (Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.)

The ability to quantify and visualize submicrometer-scale oscillatory motions of objects in three dimensions has a wide range of application in acoustics, materials sciences, and medical imaging. Here we demonstrate that volumetric snapshots of rapid periodic motion can be captured using optical coherence tomography (OCT) with subnanometer-scale motion sensitivity and microsecond-scale temporal resolution. This technique, termed OCT vibrography, was applied to generate time-resolved volumetric vibrographs of a miniature drum driven acoustically at several kilohertz.

Objectives: Minimizing collateral thermal damage during endoscopic laryngeal laser surgery remains a priority, and tissue cooling is one way to achieve this goal. Cooling systems utilizing compressed air have been shown to reduce the extent of thermal trauma on the vocal folds, but these units are not ideal for endoscopic applications because cooling is inefficient at the low airflows needed. We examined whether a novel vortex cooling device that generates cooled air at low flow rates would provide a cooling benefit beyond that which could be obtained by using room-temperature air for cooling tissue or by using no cooling during simulated laryngeal laser surgery., Methods: A continuous-wave thulium laser was used to incise glottic tissue in 12 calf vocal folds. Cooling was achieved with a prototype vortex cooler (9 degrees C air output; flow rate, 3 L/min), and tissue temperature measurements were compared to those with room-air cooling and no cooling. Thermal damage was analyzed histologically by measuring the depth of lactate dehydrogenase inactivation surrounding the mucosal incision. The cooling conditions were tested during time-constant cuts (8 seconds) and depth-constant cuts (into the thyroarytenoid muscle)., Results: During time-constant cuts, comparison between vortex cooling and room-air cooling revealed that vortex cooling resulted in a thermal damage zone that was 14% smaller (519 versus 603 microm; p < 0.05). During depth-constant cuts, vortex cooling created a thermal damage zone that was 32% smaller than that created with no cooling (p <0.01) and 9% smaller than that created with room-air cooling (p < 0.01)., Conclusions: Vortex cooling reduces thermal damage more effectively than room-air cooling or no cooling during both time-constant and depth-constant thulium laser cuts.

Objectives/hypothesis: Early glottic cancer has been involuted by treatment with the 532 nm pulsed potassium-titanyl-phosphate (KTP) laser in initial clinical studies. Selective photoangiolysis of the sublesional circulation that allows for relative sparing of surrounding tissue is the presumed mechanism. No prior controlled animal-model study has analyzed the ability of selective coagulation of lesional microvasculature coagulation with the KTP laser to involute malignant lesions. This study tests the efficacy of photoangiolysis with the KTP laser in treating squamous cell carcinoma in an established animal model., Study Design: In vivo., Methods: Malignant lesions were induced unilaterally in the cheek pouches of 21 hamsters by applying 9,10-dimethyl-1,2-benzanthrancene. The contralateral cheek pouch served as a control. Weekly lesion photodocumentation and pulsed KTP laser (30 W, 15 msec pulse width, 2 pulses/sec) treatments were done. The endpoint of each treatment was a uniform white-blanching of the lesion. Hamsters were sacrificed 1 week after the last treatment and cheek pouches were analyzed histologically., Results: Carcinoma was confirmed in 19 hamsters, and lesions that were initially <2 mm were more effectively treated than lesions that were >2 mm (P = .0004). Every lesion (10/10) that initially measured <2 mm resolved completely after laser treatment with minimal scarring noted at the treatment site based on histology. Lesions measuring between 2 and 5 mm resolved 33% of the time (2/6), and none (0/3) of the lesions >5 mm resolved after completion of the treatment period., Conclusions: Pulsed KTP laser photoangiolysis can effectively involute small malignant lesions, but may be less effective at involuting larger (>2 mm) lesions., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives: Most cases of irresolvable hoarseness are due to deficiencies in the pliability and volume of the superficial lamina propria of the phonatory mucosa. By using a US Food and Drug Administration-approved polymer, polyethylene glycol (PEG), we created a novel hydrogel (PEG30) and investigated its effects on multiple vocal fold structural and functional parameters., Methods: We injected PEG30 unilaterally into 16 normal canine vocal folds with survival times of 1 to 4 months. High-speed videos of vocal fold vibration, induced by intratracheal airflow, and phonation threshold pressures were recorded at 4 time points per subject. Three-dimensional reconstruction analysis of 11.7 T magnetic resonance images and histologic analysis identified 3 cases wherein PEG30 injections were the most superficial, so as to maximally impact vibratory function. These cases were subjected to in-depth analyses., Results: High-speed video analysis of the 3 selected cases showed minimal to no reduction in the maximum vibratory amplitudes of vocal folds injected with PEG30 compared to the non-injected, contralateral vocal fold. All PEG30-injected vocal folds displayed mucosal wave activity with low average phonation threshold pressures. No significant inflammation was observed on microlaryngoscopic examination. Magnetic resonance imaging and histologic analyses revealed time-dependent resorption of the PEG30 hydrogel by phagocytosis with minimal tissue reaction or fibrosis., Conclusions: The PEG30 hydrogel is a promising biocompatible candidate biomaterial to restore form and function to deficient phonatory mucosa, while not mechanically impeding residual endogenous superficial lamina propria.

Quantitative cross-sectional imaging of vocal folds during phonation is potentially useful for diagnosis and treatments of laryngeal disorders. Optical coherence tomography (OCT) is a powerful technique, but its relatively low frame rates makes it challenging to visualize rapidly vibrating tissues. Here, we demonstrate a novel method based on triggered laser scanning to capture 4-dimensional (4D) images of samples in motu at audio frequencies over 100 Hz. As proof-of-concept experiments, we applied this technique to imaging the oscillations of biopolymer gels on acoustic vibrators and aerodynamically driven vibrations of the vocal fold in an ex vivo calf larynx model. Our results suggest that triggered 4D OCT may be useful in understanding and assessing the function of vocal folds and developing novel treatments in research and clinical settings.

Objectives/hypothesis: Optical coherence tomography (OCT) can provide high-resolution ( approximately 10-15 microm/pixel) images of vocal fold microanatomy, as demonstrated previously. We explored physiologically triggered Fourier-domain OCT for imaging vocal folds during phonation. The goal is to visualize dynamic histological cross sections and four-dimensional data sets where multiple planes are displayed in synchronized motion. If feasible, this approach could be a useful research tool and spur development of new clinical instrumentation., Study Design: A Fourier-domain, triggered OCT system was created and tested in experiments on excised calf larynges to obtain preliminary observations and characterize important factors affecting image quality., Methods: Larynges were imaged during phonation driven by warm, humidified air. A subglottal pressure signal was used to synchronize the OCT system with the phonatory cycle. Image sequences were recorded as functions of anatomical location or subglottal pressure. Implant materials were also imaged during vibration, both in isolation and after injection into a vocal fold., Results: Oscillations of epithelium and lamina propria were observed, and parameters such as shape, amplitude, and velocity of the vocal fold mucosal waves were found to be measurable. Ripples of mucosal wave as small as 100 microm in vertical height were clearly visible. Internal strain was also observed in normal and implanted vocal folds., Conclusions: Four-dimensional OCT of the vocal fold may help to more directly relate biomechanics to anatomy and disease. It may also be useful for assaying the functional rheology of implants in the context of real tissue. With further development, this technology has potential for clinical endoscopic application.

Regenerative therapy using stem cells for the treatment of vocal fold wound healing and fibrosis is a very active area of research in Otolaryngology. Although modern phonosurgical methods can deal with many types of vocal fold pathology, vocal fold scar remains a clinical challenge. Trauma (e.g. vocal abuse, phonosurgery) and inflammation (e.g. laryngitis) are the two main causes of the vocal fold scarring. Several recent reviews detail the problem of vocal fold scarring and the array of possible solutions under investigation. The search for solutions includes autologous tissues, biomaterial implants, growth factors, anti-fibrotic agents and stem cells. This review focuses on emerging research on stem cells for vocal fold regeneration and our own studies of interactions between adipose-derived stem/stromal cells and vocal fold fibroblasts using an in vitro model. While clearly an opportunity, the challenging approach of treating vocal scarring using ASCs has just started. For future in vivo studies, improvements in cell viability and markers of stem-cell differentiation into normal fibroblasts are needed. The roles of stem cell-derived cytokines in paracrine signaling need to be further explored at a cellular level in vitro, and then extended to in vivo experiments.

Low-intensity ultrasound (LIUS) treatment has been shown to increase mass transport, which could benefit tissue grafts during the immediate postimplant period, when blood supply to the implanted tissue is suboptimal. In this in vitro study, we investigated effects of LIUS stimulation on dye diffusion, proliferation, metabolism, and tropomyosin expression of muscle cells (C2C12) and on tissue viability and gene expression of human adipose tissue organoids. We found that LIUS increased dye diffusion within adjacent tissue culture wells and caused anisotropic diffusion patterns. This effect was confirmed by a hydrophone measurement resulting in acoustic pressure 150-341 Pa in wells. Cellular studies showed that LIUS significantly increased proliferation, metabolic activity, and expression of tropomyosin. Adipose tissue treated with LIUS showed significantly increased metabolic activity and the cells had similar morphology to normal unilocular adipocytes. Gene analysis showed that tumor necrosis factor-alpha expression (a marker for tissue damage) was significantly lower for stimulated organoids than for control groups. Our data suggests that LIUS could be a useful modality for improving graft survival in vivo.

Injectable hydrogels may potentially be used for augmentation/regeneration of the lamina propria of vocal fold tissue. In this study, hyaluronic acid (HA) and dextran were chemically modified and subsequently crosslinked via formation of hydrazone bonds in phosphate buffer. Swelling ratios, degradation, and compressive moduli of the resulting hydrogels were investigated. It was found that the properties of HA-dextran hydrogels were variable and the trend of variation could be correlated with the hydrogel composition. The biocompatibility of three injectable HA-dextran hydrogels with different crosslinking density was assessed in the vocal fold region using a ferret model. It was found that HA-dextran hydrogels implanted for three weeks stimulated mild foreign-body reactions. Distinct tissue-material interactions were also observed for hydrogels made from different formulations: the hydrogel with the lowest crosslinking density was completely degraded in vivo; while material residues were visible for other types of hydrogel injections, with or without cell penetration into the implantation depending on the hydrogel composition. The in vivo results suggest that the HA-dextran hydrogel matrices can be further developed for applications of vocal fold tissue restoration., ((c) 2010 Wiley Periodicals, Inc.)

Adipose-derived stem cells (ASCs) may provide a clinical option for rebuilding damaged superficial lamina propria of the vocal fold. We investigated the effects of five hydrogels (hyaluronic acid [HA], collagen, fibrin, and cogels of fibrin-collagen and fibrin-HA) on the differentiation of ASCs, with the long-term goal of establishing the conditions necessary for controlling the differentiation of ASC into the functional equivalent of superficial lamina propria fibroblasts. Human ASCs were isolated and characterized by fluorescence-activated cell sorting and real-time polymerase chain reaction. According to fluorescence-activated cell sorting and gene analysis, over 90% of isolated ASCs expressed adult stem cell surface markers and expressed adult stem cell genes. Scaffold-specific gene expression and morphology were assessed by culturing the ASCs in three-dimensional hydrogels. Twofold higher amounts of total DNA were detected in fibrin and cogel cultures than in collagen and HA cultures. Elastin expression was significantly higher in cells grown in fibrin-based gels than in cells grown in other gels. Cells grown in the cogels showed elongated morphology, expressed decorin marker, and exhibited glycosaminoglycan synthesis, which indicate ASC differentiation. Our data suggest that it may be possible to control the differentiation of ASCs using scaffolds appropriate for vocal fold tissue engineering applications. In particular, cogels of HA or collagen with fibrin enhanced proliferation, differentiation, and elastin expression.

Objectives/hypothesis: To explore whether adipose-derived stem/stromal cells (ASCs) have therapeutic potential for treating scarred superficial lamina propria through the effects of secreted hepatocyte growth factor (HGF) on scar fibroblasts., Study Design: In vitro study using coculture system., Methods: Scar fibroblasts (SFs) were isolated from ferret vocal folds electrocauterized 2 weeks previously. ASCs were isolated from ferret lipoaspirated subcutaneous abdominal fat. For coculture experiments, the two cell types were combined in Transwell plates for 6 days, followed by 1 or 3 days of monoculture after removing the upper chamber. Assays were then performed on cells and media from the bottom chamber. We measured: 1) the production of hyaluronic acid (HA), collagen and HGF via enzyme-linked immunosorbent assays, 2) the expression of alpha-smooth muscle actin (alpha-SMA), 3) cell proliferation, and 4) apoptosis of SFs (2, 3, and 4 via flow cytometry). Other experiments examined the effects of HGF on SFs and the effects of HGF neutralization in the coculture system., Results: Coculture led to significant decreases in SF collagen production (P < .05), cell proliferation (P < .05), and alpha-SMA expression (P < .05), whereas HA production increased (P < .05). Coculture also increased HGF secretion from ASCs (P < .05). Neutralization of HGF abolished the inhibitory effects of ASCs on SF collagen synthesis (P < .05)., Conclusions: ASCs influence SFs to adopt a less fibrotic profile. It appears that HGF is at least one of the soluble factors responsible for this effect. Implanted ASCs could potentially ameliorate vocal fold scar by acting as long-term, intrinsic sources of HGF.

Objectives/hypothesis: Optical coherence tomography (OCT) is a promising new imaging modality that can help discern the layered microstructure of vocal folds. In the future, subepithelial injections of implants will improve vocal fold pliability where there is stiffness of phonatory mucosa. Using OCT both to delineate the depth of subepithelial injections real-time and to serially image the implant over time would be valuable, and has not been demonstrated previously., Study Design: Ex vivo study using excised calf larynges and survival study using canines in vivo., Methods: An investigation was done employing real-time OCT imaging of subepithelial injection pulses into phonatory mucosa of four calf larynges ex vivo to track the presence of subepithelial implants in phonatory mucosa in a survival study using an in vivo canine model., Results: OCT readily identified polyethylene glycol (PEG)-based hydrogel in the subepithelial plane in both the ex vivo calf study and the in vivo canine study. Ex vivo calf images correlated with histological specimens obtained immediately postinjection. Images obtained in this study provide confirmation of the hydrogel injection depth in real time, and allow for the implant to be tracked during a canine survival study., Conclusions: OCT can confirm subepithelial placement of hydrogel implant in the vocal fold with sufficient resolution to provide instantaneous feedback of an injection pulse. Survival studies in an in vivo canine model indicate that OCT can potentially be helpful in monitoring rheologically appropriate implants within the superficial lamina propria.

Objectives/hypothesis: To explore adipose-derived stem cell/fibroblast interactions as a potential remodeling pathway for vocal fold scar., Study Design: Fibroblasts and adipose-derived stem/stromal cells (ASCs) were cultured alone and in combination in a cell-contact-independent paracrine system. Analyses of cell proliferation, and the production of hyaluronic acid (HA) and collagen were performed on samples collected on days 1, 3, and 7., Methods: Normal fibroblasts (NFs) were isolated bilaterally from the subepithelial lamina propria of two normal ferret vocal folds. Scar fibroblasts (SFs) were isolated from vocal folds that were electrocauterized 2 weeks before harvest. ASCs were isolated from lipoaspirated subcutaneous abdominal fat of two ferrets. A transwell cell-contact-independent cell communication culturing system was used for coculture experiments. Cells were seeded at 50,000/well in both monoculture and coculture experiments., Results: In monoculture, SFs proliferated faster and produced less HA and more collagen than NFs at day 7 (P < .05). In SF/ASC coculture, SF proliferation was diminished and collagen production at day 7 decreased (P < .05). HA production did not differ between monoculture and coculture conditions., Conclusions: Normal and scar-tissue-derived vocal fold fibroblasts maintain phenotypic differences in culture, thus validating this in vitro scar model. In co-culture, contact-independent crosstalk occurs between SFs and ASCa, leading to less collagen secretion. The data support the hypothesis that ASCs can induce favorable remodeling of scarred vocal folds in vivo by their interactions with endogenous fibroblasts.

Objectives/hypothesis: High-frequency ultrasound imaging offers the potential for assisting in the diagnosis and treatment of vocal fold pathology if it allows aspects of vocal fold microstructure to be visualized noninvasively. The objective of this study was to assess the ability of high-frequency ultrasound to image vocal fold anatomy and injected biomaterials., Study Design: The vocal folds of two excised calf larynges were imaged ex vivo and compared with corresponding histological sections., Methods: High-frequency ultrasound imaging was performed under saline submersion using 40 and 50 MHz transducers, and corresponding cryostat cross-sections were stained with H&E, Trichome, and Verhoeff's Van Gieson stains., Results: The epithelial surface, lamina propria, and underlying muscle were easily identified with the high-frequency ultrasound as verified with histological sections representing each imaged region. The arytenoid cartilage vocal process can also be clearly distinguished from the surrounding tissue, as can the full extent of injected biomaterials within the superficial lamina propria. Useful ultrasound resolution was obtained to depths of at least 10 mm within the tissue with the 40 MHz transducer., Conclusions: This preliminary study demonstrates the capability of high-frequency ultrasound to image the layered anatomy of the calf vocal fold and to discern materials injected into the superficial lamina propria, indicating that this technology holds a strong potential for use in phonosurgery.

Objectives/hypothesis: The optimal balance between a laser's clinical efficacy and collateral thermal damage is the major determinant for selection of a particular laser in endolaryngeal surgery. The chick chorioallantoic membrane (CAM) simulates the microvasculature of the human vocal fold and is, therefore, useful for testing effects of laser settings, mode of delivery, active cooling, and wavelength. Such information is essential for optimizing the effectiveness of lasers in treating laryngeal pathology while preserving vocal function., Study Design and Methods: The thermal and coagulative effects of four lasers (585 nm PDL, 532 nm KTP, 2.01ìm Thulium, 10.6ìm CO2) were quantified at selected (and clinically relevant) energy settings before and after tissue cooling using the CAM model. Measures included imaging real-time vascular reactions in the CAM model (i.e., vessel coagulation and/or rupture), and post-procedure histologic analysis of CAM tissue. In each experiment, laser energy was applied to the CAM in a controlled manner. Cooling was done using a dermatological cold-air device, and temperatures were measured with a thermistor. Lasers tested included the photoangiolytic pulsed-dye (PDL) and KTP, as well as the ablative/cutting CO2 and thulium lasers. The vessel rupture/coagulation and thermal effects of various energy-delivery parameters on the CAM, with and without cooling, were assessed. After removal of the CAM, specimens were stained as whole-mounts, photographed at 4X magnification, and evaluated by two independent, blinded surgeon reviewers. The efficacy of increased pulse-width (KTP laser) on treating larger vessels (>0.5 mm) and the effects of extravasated blood on photoangiolysis were also evaluated., Results: Photoangiolytic lasers: Vessel coagulation/rupture rates showed that the PDL caused more frequent vessel rupture than the KTP laser. For both lasers, cooling the CAM by approximately 20 degrees C resulted in 30% - 60% reduction in the thermal-damage zone (P < .05). Cooling reduced the efficacy of coagulation with the PDL but not with the KTP laser. The clinically observed phenomenon that laser heating of extravasated blood increases thermal damage and decreases efficacy of coagulation was clearly evident in the CAM model. Ablative lasers: The thermal-damage zone of the CO2 laser (0.3 mm spot size) was not significantly different with or without cooling (0.32 mm2 and 0.34 mm2, respectively) (P = .30). However, when the spot size was defocused to 1 mm, the thermal-damage zone was over 2x greater when the tissue was not cooled (0.74 mm2 vs 0.35 mm2) (P < .002). The thermal-damage zone of the Thulium laser was reduced by an average of 58% for the three power settings tested when the CAM was air-cooled (P < .05)., Conclusions: The CAM was an excellent model for studying the effects of photoangiolytic lasers, for which optimal pulse-widths exist for vessel coagulation. Smaller vessels coagulated reliably at pulse widths >15 msec, and larger vessels required pulse widths >35 msec for optimal coagulation. Cooling the target tissue decreased the thermal-damage zone created by photoangiolytic lasers. While cooling had no effect on the efficacy of coagulation with longer pulse widths (KTP), tissue cooling decreased the coagulation rate at shorter pulse widths (PDL). The thermal effects of cutting/ablating lasers can be reduced with cooling, but the CAM was not a good model with which to study coagulation/rupture rates in cutting/ablating lasers.

Controlling and guiding cell behavior requires scaffolding materials capable of programming the three-dimensional (3-D) extracellular environment. In this study, we devised a new self-assembling peptide template for synthesizing nanofibrous hydrogels containing cell-responsive ligands. In particular, the insertion of a matrix metalloproteinase-2 (MMP-2) labile hexapeptide into the self-assembling building blocks of arginine-alanine-aspartate-alanine (RADA) was investigated. A series of peptides, varied by the position of the MMP-2 hexapeptide substrate and the length of RADA blocks, were prepared by parallel synthesis. Their self-assembling capabilities were characterized and compared by circular dichroism spectroscopy and dynamical mechanical analysis. Among all the different insertion patterns, the sequence comprising a centrically positioned MMP-2 substrate was flanked with three RADA units on each side self-assembled into a hydrogel matrix, with mechanical properties and nanofiber morphology comparable to the native material built with (RADA)(4) alone. Exposure of the new gel to MMP-2 resulted in peptide cleavage, as confirmed by mass spectroscopy, and a decrease in surface hardness, as detected by nanoindentor, indicating that the enzyme mediated degradation was localized to the gel surface. The new design can be used for introducing biological functions into self-assembling peptides to create scaffolding materials with potential applications in areas such as tissue engineering and regenerative medicine.

Objectives: Evolving photoangiolytic laser techniques for treating vocal fold lesions motivated the development of a model for research and surgical training. The chick chorioallantoic membrane (CAM), which is composed of a microvasculature suspended within the egg albumen, simulates the vocal fold microcirculation within the superficial lamina propria (SLP). To characterize this model, we compared measurements of vessel diameters to superficial vessels in human vocal folds., Methods: The diameters of first-, second-, and third-order CAM vessels were measured in fertilized chicken eggs. The superficial blood vessels of the human vocal fold were measured from intraoperative images., Results: According to the branching pattern, vessel segments were identified as first-, second-, or third-order, with average diameters of 0.035 mm (0.02 to 0.1 mm), 0.18 mm (0.12 to 0.41 mm), and 0.8 mm (0.6 to 0.98 mm), respectively. The total vessels measured included 362 first-order, 119 second-order, and 82 third-order vessels. In 10 adult human vocal folds, an average vessel diameter of 0.04 mm (0.015 to 0.1 mm) was observed in 50 vessels., Conclusions: The CAM microvasculature suspended in albumen provides a useful surgical model simulating the microcirculation within the SLP of the human vocal fold. Although first-order CAM vessels best approximate the size of normal vocal fold subepithelial vessels seen at surgery, second- and third-order vessels resemble the vascular abnormalities frequently encountered during microsurgery for phonotraumatic lesions.

Objectives: The 2-microm-wavelength thulium laser has recently been shown to be an effective cutting instrument in endolaryngeal surgery, although there is increased thermal trauma as compared with the carbon dioxide laser. This study investigated temperature changes and thermal trauma during thulium laser dissection of laryngeal tissue, with and without air cooling, in an ex vivo model., Methods: A continuous-wave thulium laser (400-microm fiber, 4-W continuous power, 4-second duration) was used to incise 10 calf vocal folds. Paired cooled and uncooled cuts were made in each fold with a dermatologic cooling device. A thermistor inserted into the glottic subepithelium was used to measure tissue temperatures. Thermal damage was analyzed histologically by measuring the depth of the zone of lactate dehydrogenase inactivation surrounding the mucosal incision., Results: The initial vocal fold temperature averaged 24.3 degrees C without cooling and 4.4 degrees C with cooling. The peak temperature during cutting averaged 59.1 degrees C without cooling and 28.0 degrees C with cooling. The thermal damage zone surrounding the cooled incisions was approximately 27% less than that surrounding the uncooled incisions., Conclusions: Air cooling can reduce the extent of thermal trauma associated with thulium laser surgery of the vocal folds, and the high-temperature plume generated during laser cutting is effectively cleared.

Quantification of the elastic properties of the human vocal fold provides invaluable data for researchers deriving mathematical models of phonation, developing tissue engineering therapies, and as normative data for comparison between healthy and scarred tissue. This study measured the shear modulus of excised cadaver vocal folds from 20 subjects. Twenty freshly excised human larynxes were evaluated less than four days post-mortem. They were split along the saggital plane and mounted without tension. Shear modulus was obtained by two different methods. For method 1 cyclical shear stress was applied transversely to the mid-membranous portion of the vocal fold, and shear modulus derived by applying a simple shear model. For method 2 the apparatus was configured as an indentometer, and shear modulus obtained from the stress/strain data by applying an established analytical technique. Method 1 shear model for male larynxes yielded a range from 246 to 3,356 Pa, with a mean value of 1,008 and SD of 380. The range for female larynxes was 286-3,332 Pa, with a mean value of 1,237 and SD of 768. Method 2 indentometer model for male larynxes yielded a range from 552 to 2,741 Pa, with a mean value of 1,000 and SD of 460. The range for female larynxes was 509-1,989 Pa, with a mean value of 1,332 and SD of 428. We have successfully demonstrated two methodologies that are capable of directly measuring the shear modulus of the human vocal fold, without dissecting out the vocal fold cover tissue. The sample size of nine female and 11 male larynxes is too small to validate a general conclusion. The high degree of variability in this small cohort of subjects indicates that factors such as age, health status, and post-mortem delay may be significant; and that there is range of 'normality' for vocal fold tissue.