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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.

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

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.

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.Laboratory study.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.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.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.NA Laryngoscope, 129:E229-E237, 2019.

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 R2 > 0.98) between the readout and direct measures of volume, even for small volumes (

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 (P = .0004). Every lesion (10/10) that initially measured 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.

Objectives/Hypothesis: Optical coherence tomography (OCT) can provide high-resolution (! 10‐15 lm/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 lm 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.

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 healuronic acid (HA), collagen and HGF via enzymelinked immunosorbent assays, 2) the expression of α-smooth muscle actin (α-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 α-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: High-resolution imaging of vocal folds that distinguishes vocal fold (VF) layered microstructure and VF implants would provide a key experimental tool for translational research investigating biomaterial-based interventions to treat vocal fold scar. To establish proof of concept, we studied whether 11.7 Tesla (T) magnetic resonance (MR) microimaging provides the needed resolution to resolve vocal fold tissue architecture. Study Design: We performed ex vivo MR microimaging of fixed ferret and canine larynges to determine whether changes in the layered architecture can be detected in the presence of scar and subsequent to biomaterial injections into the vocal folds. Serial section histological analyses were done to corroborate MR microimaging findings. Methods: Multiple axial and transverse/coronal 300-μm slices were obtained using an 11.7 T MR spectrometer/500 MHz for proton with gradient-recalled echo and rapid acquisition with relaxation enhancement imaging sequences. Results: High-resolution (39 μm/pixel) MR microimages distinguished VF epithelium, lamina propria, muscle, and cartilage in ferret and canine larynges. In ferret scarred VFs (n = 25), collagen-rich dense scar tissue was distinguishable from contralateral nonscarred VFs and from normal ferret VFs (n = 25), as confirmed on histology. MR microimaging accurately detected injected autologous fat, hyaluronic acid-based and polyethylene glycol (PEG)-based implants injected into both ferret and canine VFs. Importantly, MRI accurately showed resorption of PEG implants in ferrets and canines, as confirmed on histology. Additionally, ex vivo MR spectroscopy distinguished fat from PEG-based implants. Conclusions: Ex vivo 11.7 T MR microimaging provided high-resolution images of ferret and canine laryngeal tissue microstructure, although the superficial lamina propria could not be distinguished. Histology confirmed MR microimaging findings, indicating utility of MR microimaging of modeled scar, implant residence time, and tissue responses, thus providing integrative insight relevant to translational research. Laryngoscope, 2009

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. © 2009 The American Laryngological, Rhinological and Otological Society, Inc.

Objectives: Selective vascular ablation (photoangiolysis) using pulsed lasers that target hemoglobin is an effective treatment strategy for many vocal fold lesions. However, vessel rupture with extravasation of blood reduces selectivity for vessels, which is frequently observed with the 0.45-ms, 585-nm pulsed dye laser. Previous studies have shown that vessel rupture is the result of vaporization of blood, an event that varies with laser pulse width and pulse fluence (energy per unit area). Clinical observations using a 532-nm wavelength pulsed potassium-titanyl-phosphate (KTP) laser revealed less laser-induced hemorrhage than the pulsed dye laser. This study investigated settings for the pulsed KTP laser to achieve selective vessel destruction without rupture using the avian chorioallantoic membrane under conditions similar to flexible laryngoscopic delivery of the laser in clinical practice. Study Design: The chick chorioallantoic membrane offers convenient access to many small blood vessels similar in size to those targeted in human vocal fold. Using a 532-nm pulsed KTP laser, pulse width, pulse energy, and working distance from the optical delivery fiber were varied to assess influence on the ability to achieve vessel coagulation without vessel wall rupture. Methods: Thirdorder vessels (n = 135) were irradiated: Energy (471-550 mJ), pulse width (10, 15, 30 ms), and fiber-to-tissue distance (1 mm, 3 mm) were varied systematically. Results: Selective vessel destruction without vessel wall rupture was more often achieved by increasing pulse width, increasing the fiber-to-tissue distance, and decreasing energy. Vessel destruction without rupture was consistently achieved using 15- or 30-ms pulses with a fiber-to-tissue distance of 3 mm (pulse fluence of 13-16 J/cm 2 ). Conclusions: This study substantiates our clinical observation that a 532-nm pulsed KTP laser was effective for ablating microcirculation while minimizing vessel wall rupture and hemorrhage.

Objectives: Intraoperative assessment of vocal fold vibration during phonomicrosurgery performed under general anesthesia may enhance surgical decision-making. We therefore developed and bench-tested a new device we refer to as the aerodynamic vocal fold driver (AVFD). Materials and Methods: The AVFD comprises a hand-held probe that uses airflow to drive individual vocal folds into phonatory-like vibration. This permits stroboscopic visualization of mucosal waves with simultaneous control of subglottal air pressure. In initial experiments to validate the technique, AVFD driven phonation and conventional whole-larynx phonation were compared using excised canine larynges (n = 14). Results: Single vocal fold phonation using the AVFD and whole larynx phonation yielded similar, positive correlations between subglottal pressure and both amplitude and frequency of vibration. Experiments simulating vocal fold scar-related mucosal stiffening by subepithelial injection of fixative showed the expected elevation of phonation threshold pressures as measured with the AVFD. Likewise, unilateral tissue compression injury disrupted vocal fold vibration, and the AVFD was useful for quantifying improvement in the damaged vocal fold after repair with injection of cross-linked hyaluronic acid gel. Conclusions: These results show that this new instrument has the potential to provide novel and useful information for laryngeal experimentation and to improve phonosurgery.

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.In vivo.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.Carcinoma was confirmed in 19 hamsters, and lesions that were initially2 mm were more effectively treated than lesions that were2 mm (P = .0004). Every lesion (10/10) that initially measured2 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 lesions5 mm resolved after completion of the treatment period.Pulsed KTP laser photoangiolysis can effectively involute small malignant lesions, but may be less effective at involuting larger (2 mm) lesions.

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.In vitro study using coculture system.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.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).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.

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.Ex vivo study using excised calf larynges and survival study using canines in vivo.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.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.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

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 highfrequency 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: Fat preparation for vocal-fold injection medialization is typically done by scraping of excised fat or by lipo-aspiration; however, lipo-aspiration is substantially more efficient. Considering this, we compared viability of fat tissues obtained by these two techniques. We also examined whether basic fibroblast growth factor (bFGF) would increase cell proliferation in samples harvested by these methods. Study Design: Harvesting techniques (scraping and lipo-aspiration) were compared using both human and ferret fat. In vitro assays were used to assess tissue viability and cell proliferation. Methods: Human (n = 5) and ferret (n = 15) abdominal fat specimens were harvested by scraping and lipo-aspiration, for a total of 40 specimens. Alamar Blue and glycerol-3-phosphate dehydrogenase assays were used to quantitatively assess metabolic activity and cellular damage immediately after harvest. PicoGreen assays assessed cell proliferation by quantifying total DNA in harvested specimens after 0, 14, or 21 days in culture. The effects of bFGF (10 ng/mL) on proliferation were measured for the same timepoints. Results: The glycerol-3-phosphate dehydrogenase assay indicated that lipo-aspiration caused more initial tissue damage (12 ± 5 mU/mL) than scraping (5 ± 3 mU/mL), but cell metabolic activity was similar in both groups based on the Alamar Blue assay. Cell proliferation at 14 and 21 days was significantly higher for lipo-aspirated fat than for scraped fat (92.5 ± 8.8 vs. 55.1 ± 1.3 ng DNA at 14 days and 111.1 ± 10.5 vs. 44.6 ± 4.1 ng DNA at 21 days). bFGF increased fibroblast-like cell proliferation significantly for both harvesting methods at day 21. Conclusions: Lipo-aspiration caused more initial damage than scraping, but may yield better long-term viability based on increased proliferation. bFGF may enhance cellularity of the stromal component grafted adipose tissue.

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, 2.01im Thulium, 10.6im 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 ∼20°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 2× 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.

Objectives/Hypothesis: Whereas vibration of the vocal folds has been analyzed and modeled in detail, less attention has been paid to quantifying the slower motions of abduction and adduction. Because these gestures reflect neuromuscular function, cartilage geometry, and joint mobility, objective measurement might improve assessment and treatment of patients with neuromuscular or structural disease. Study Design: Prospective, observational. Methods: Twenty-one normal adult volunteers performed a repeated “ee-sniff” task at three rates during flexible endoscopy. Distortion in the images was corrected and frame-by-frame analysis of the angle between the true vocal folds (glottic angle) yielded measures of maximum abduction angle (MAA) and mean angular velocities of abduction (MVAB) and adduction (MVAD). Results: Reliability tests indicated interjudge variability of 3.8 degrees (deg). The mean MAA was 51 deg and ranged from 31 to 77 deg across subjects. The mean MVADs were 229, 309, and 475 deg/s for slow, medium, and fast rates, respectively, and the mean MVABs were 330, 388, and 441 deg/s for slow, medium, and fast rates, respectively. Statistical analysis showed greater influence of gesture rate on adduction than on abduction. Conclusions: The glottic angle can be reliably measured from flexible endoscopic images and angular velocities of vocal fold abduction and adduction can be determined from analysis of sequential video frames. Accuracy is significantly enhanced by correction of distortion in the images. This approach holds promise for objective characterization of vocal fold function in patients with a variety of disorders.

Objectives: To introduce a new tool for phonosurgical training and education. A multitude of innovations in complex laryngeal surgery has catalyzed new educational initiatives. Establishing dexterity in phonomicrosurgery is often difficult to achieve while working on patients because of the narrow margin for success. Furthermore, laryngoplastic phonosurgery and open partial laryngectomy require sophisticated knowledge of precise anatomic relationships, which can be difficult to express in images. Finally, many teaching programs do not have a high volume of these procedures, and there is a significant need to transmit this information in continuing education courses. Study Design: Prototype design. Methods: A laryngeal dissection station (LDS) was designed to facilitate the acquisition of high-level procedural skill sets for both transoral and transcervical techniques. Results: This LDS can be used in existing temporal-bone laboratories by using cadaveric larynges. A rectangular frame supports two adjustable holders, one for the larynx and one for the examining speculum of a laryngoscope. Procedures are performed with the larynx fixed in space by a novel fixator. Variation in position and orientation of the components affords simulation of both microlaryngoscopy and open surgery. The dissection station can accommodate virtually any laryngoscope, regardless of size or shape. Conclusions: This training apparatus should facilitate laryngeal surgical instruction in residency training and continuing medical education. This device and others like it can help establish clinical competency in laryngology, should this become necessary in future educational models of residency training and recertification.

Objectives/Hypothesis: Because current tongue reconstructive methods introduce adynamic, variably sensate tissue into the mouth, the critical functions of the tongue in articulation and deglutition may be compromised. The objective of this work was to introduce a combination of myoblasts and scaffolding material into rat hemiglossectomy defects and to examine the extent of neomuscle formation in the reconstructed area, under the hypothesis that the presence of myoblasts leads to formation of new muscle. Study Design: Randomized, prospective animal study. Methods: Myoblasts were harvested from neonatal Lewis rats, and a growth factor enriched collagen gel was prepared. Syngeneic adult animals received either hemiglossectomy alone or reconstruction with one of four experimental reconstructive preparations: collagen gel alone, collagen gel with suspended myoblasts, the gel-cell combination in undifferentiated muscle construct form by way of tissue culture for 7 days in a preformed mold, or differentiated constructs, cultured in myoblast fusion medium. After 6 or 16 weeks, animal weight gain was recorded, animals were killed, and the tongues harvested. The tissue was examined histologically, and quality of the muscular regenerate was rated on a scale according to predefined criteria. Results: Animals in all groups gained weight appropriately. In groups receiving hemiglossectomy alone or acellular (gel only) reconstruction, there was significant scarring and lack of neomuscle formation. In groups receiving myoblast transplantation, either by way of gel suspension or in the form of undifferentiated or differentiated constructs, muscle quality was superior to controls. Conclusions: Myoblast transplantation into hemiglossectomy defects appears to lead to new muscle formation and does not inhibit normal weight gain in animals after tongue implantation.