Functional and Histological Evaluation following Canine Vocal Fold Reconstruction Using Composite Thyroid Ala Perichondrium Flaps.

OBJECTIVES/HYPOTHESES: We evaluated the effects of vocal fold reconstruction using a composite thyroid ala perichondrium flap (CTAP) after unilateral vocal fold stripping in beagles. We hypothesized that CTAP would improve glottic closure, decrease phonation threshold pressure, and decrease perturbation. In addition, vocal folds with CTAP would exhibit neovascularization and fat with increased von Willebrand factor (vWF) and smooth muscle actin (SMA), reflecting neoangiogenesis and flap viability. STUDY DESIGN: Randomized controlled trial using beagles. SETTING: University laboratory. METHODS: Ten beagles underwent unilateral vocal fold stripping. Dogs in the scar-only group (n = 5) were sacrificed at 1 month. Dogs in the CTAP group (n = 5) underwent ipsilateral reconstruction with CTAP at 1 month and were sacrificed at 2 months. Excised larynx experiments evaluated vocal fold vibration using aerodynamic, acoustic, and mucosal wave measurements. Qualitative evaluation of vocal fold morphology and quantitative analysis of elastin, collagen, glycosaminoglycans, vWF, SMA, and hyaluronic acid were performed. RESULTS: Phonation threshold pressure (P = .005), percent jitter (P = .010), percent shimmer (P = .007), and open quotient (P = .007) were lower in the CTAP group. Neovascularization (P = .0079) and fat (P = .1667) occurred more with CTAP, although the difference in fat was not significant. von Willebrand factor was higher with CTAP vs contralateral normal fold (P = .110), although not statistically significant. Smooth muscle actin was higher with CTAP vs contralateral normal fold (P = .038) and scarred vocal folds (P = .022). CONCLUSIONS: Composite thyroid ala perichondrium flap restored glottic closure and vibratory periodicity following vocal fold scarring. Additional investigation on biologic response is warranted. Composite thyroid ala perichondrium flap offers an autologous, vascularized implant that can improve both vocal fold structure and function.

Stretch calculated from grip distance accurately approximates mid-specimen stretch in large elastic arteries in uniaxial tensile tests.

The mechanical properties of vascular tissues affect hemodynamics and can alter disease progression. The uniaxial tensile test is a simple and effective method for determining the stress-strain relationship in arterial tissue ex vivo. To enable calculation of strain, stretch can be measured directly with image tracking of markers on the tissue or indirectly from the distance between the grips used to hold the specimen. While the imaging technique is generally considered more accurate, it also requires more analysis, and the grip distance method is more widely used. The purpose of this study is to compare the stretch of the testing specimen calculated from the grip distance method to that obtained from the imaging method for canine descending aortas and large proximal pulmonary arteries. Our results showed a significant difference in stretch between the two methods; however, this difference was consistently less than 2%. Therefore, the grip distance method is an accurate approximation of the stretch in large elastic arteries in the uniaxial tensile test.

Elucidation of soft tissue flap histologic margins within a canine vocal fold.

BACKGROUND: Histologic identification of implanted soft tissues in experimental animal models can be challenging, as donor tissue often strongly resembles the recipient bed. We have encountered this dilemma following implantation of a Composite Thyroid Ala Perichondrium flap (CTAP) into a vocal fold. The CTAP procedure is the first to utilize a vascularized flap for vocal fold reconstruction, making data to confirm or refute its viability critical. The current study evaluated several tissue stains to define precisely the histologic margins of CTAPs at two weeks post-implantation in a canine model. METHODS: Initial testing exposed canine cadaveric tissues to four stains (tattoo ink, Congo red, 4'6-diamidino-2-phenylindole, and henna) across four time periods. Tattoo ink alone withstood histologic processing. An exposure of 1 minute adequately delineated CTAP boundaries. The study concluded with a canine in vivo evaluation of a CTAP exposed to tattoo ink for 1 minute. After a two-week recovery period, vocal folds were harvested and evaluated histologically. RESULTS: Tattoo ink proved to be a safe and effective histologic marker in vivo, where the histologic margins of the implanted CTAP were clearly demarcated by a thin band of tattoo ink, soft tissue reactions were minimal, and interference with standard, special, or immunohistochemical stain assessments did not occur. CONCLUSIONS: Tattoo ink provides a reliable means of demarcating a CTAP within a vocal fold and demonstrated that CTAPs survive transplantation. Further, tattoo ink demarcation may serve as a useful histologic marker for those wishing to assess tissue implants in other in vivo models.

Pulmonary artery relative area change is inversely related to ex vivo measured arterial elastic modulus in the canine model of acute pulmonary embolization.

A low relative area change (RAC) of the proximal pulmonary artery (PA) over the cardiac cycle is a good predictor of mortality from right ventricular failure in patients with pulmonary hypertension (PH). The relationship between RAC and local mechanical properties of arteries, which are known to stiffen in acute and chronic PH, is not clear, however. In this study, we estimated elastic moduli of three PAs (MPA, LPA and RPA: main, left and right PAs) at the physiological state using mechanical testing data and correlated these estimated elastic moduli to RAC measured in vivo with both phase-contrast magnetic resonance imaging (PC-MRI) and M-mode echocardiography (on RPA only). We did so using data from a canine model of acute PH due to embolization to assess the sensitivity of RAC to changes in elastic modulus in the absence of chronic PH-induced arterial remodeling. We found that elastic modulus increased with embolization-induced PH, presumably a consequence of increased collagen engagement, which corresponds well to decreased RAC. Furthermore, RAC was inversely related to elastic modulus. Finally, we found MRI and echocardiography yielded comparable estimates of RAC. We conclude that RAC of proximal PAs can be obtained from either MRI or echocardiography and a change in RAC indicates a change in elastic modulus of proximal PAs detectable even in the absence of chronic PH-induced arterial remodeling. The correlation between RAC and elastic modulus of proximal PAs may be useful for prognoses and to monitor the effects of therapeutic interventions in patients with PH.

Technical refinements to the minithyrotomy procedure.

OBJECTIVES/HYPOTHESIS: Although the minithyrotomy (MT) procedure was introduced in 1999, it has not been widely used for voice restoration. Its limited dissemination is due in part to lack of appropriate implants and in part due to technical challenges. The 2011 introduction of the composite thyroid ala perichondrium flap (CTAP) into a vocal fold through an MT was designed to supply an appropriate implanted tissue. However, technical difficulties persisted. Noted impediments have included limited surgical access, potential CTAP pedicle constriction during healing, lack of specialized surgical instrumentation, and potential retraction or extrusion of CTAPs. This study was performed to address these technical challenges with or without the use of CTAP reconstruction. STUDY DESIGN: Experimental. Cadaveric and in vivo canine model. METHODS: Experimentation on canine cadaveric larynges yielded MT and CTAP alterations, instrument creation, and implant affixation procedures. These refinements were applied in vivo using canine subjects. Two weeks post-CTAP repair, subjects were humanely euthanized, followed by laryngeal harvest and histologic analysis of the vocal folds. RESULTS: Refinements to CTAP modification, MT, instrumentation, and CTAP affixation are successful in vivo, just as in preliminary cadaveric trials. CONCLUSIONS: The proposed refinements were designed to improve the outcomes achieved via a CTAP specifically but have utility for any MT approach. Continued improvements to specialized instrumentation are necessary. Enhanced affixation of a CTAP, with improved accuracy and easier deployment are also essential. Further refinements should allow more reliable implementation of the MT by a growing number of laryngeal surgeons.

Local vascularized flaps for augmentation of Reinke's space.

OBJECTIVES/HYPOTHESIS: The purpose of this study is to describe and test a novel surgical strategy for augmentation of Reinke's space using vascularized flaps: a thyroid ala perichondrium flap (TAP) and a composite thyroid ala perichondrium flap (CTAP) from the anterior larynx. We hypothesized that these specially designed vascularized flaps would remain viable once inset into the lamina propria, and that they would not disrupt rheologic, biomechanical, and histologic properties of the native vocal fold. STUDY DESIGN: Experimental. In vivo canine model. METHODS: The length and volume of test flaps harvested in six adult human cadaveric larynges were analyzed to determine suitability for use in augmentation in the lamina propria. Also, 12 beagles randomly underwent unilateral placement of either TAP or CTAP, which were designed in accordance with the human adult cadaveric experiments. Flap perfusion was measured before and after harvest with laser Doppler. After 1 month, the beagles were humanely sacrificed and their larynges subjected to aerodynamic and acoustic evaluation using an excised larynx apparatus. The vocal fold lamina propria of four larynges--two TAP and two CTAP--underwent rheologic evaluation using a simple-shear rheometer. The remaining eight larynges underwent quantitative histologic and immunohistochemical evaluation. The survival and complication (swallowing, airway, local wound) rates of all dogs were noted. RESULTS: Initial studies with adult human cadaveric larynges established that TAP and CTAP possessed length and volume greater than native lamina propria. In the canine experiments, the perfusion change in the flaps was similar between flap groups. The damping ratio (ζ), dynamic viscosity (η'), elastic shear modulus (G'), and viscous shear modulus (G″) of treated and untreated native vocal folds were not statistically different. The glottic function measures of vocal efficiency, laryngeal resistance, jitter, shimmer, and harmonics-to-noise ratio (HNR) of treated and normal larynges were not statistically different. Similarly, the values for collagen, elastin, and glycosaminoglycans (GAGs) in treated and untreated vocal folds were not statistically different. Also, neither neochrondrogenesis nor neoosteogenesis was detected in any treated vocal fold. The values for vascular and cellular proliferation in treated and untreated vocal folds were not statistically different. All test dogs survived and had no complications related to swallowing, airway distress, or the local wound. CONCLUSIONS: The test flaps described and tested in this study appear to have conceptually attractive features for augmentation of Reinke's space. When placed in an in vivo setting TAP and CTAP did not reveal unfavorable vascular, rheologic, aerodynamic, acoustic, or histologic characteristics. There was no unanticipated morbidity or mortality to the test animals. Long-term viability of these flaps is unknown. TAP and CTAP may open novel pathways for correction of glottic defects and may offer crossover opportunities with tissue engineering techniques.

Gradation of stiffness of the mucosa inferior to the vocal fold.

During phonation, energy is transferred from the subglottal airflow through the air/mucosa interface that results in the propagation of the mucosal wave in the vocal fold. The vocal fold is soft, and the subglottal mucosa is stiff. We hypothesize that it is highly improbable that there is a rigid boundary between the tissue structures, with a sudden drop in stiffness; and that a gradual change would be more likely to support the efficient transfer of energy from the airflow to the mucosal wave. Our objective was to test this hypothesis by quantifying the change in mucosa stiffness with respect to anatomical position. In this initial study, using five pig larynges, a series of point-specific measurements of mucosa stiffness were taken in a line from the midpoint of the vocal fold toward the trachea. A modified linear skin rheometer adapted for laryngeal elasticity measurement applied shear stress to a series of seven positions at 2-mm intervals starting from the midmembranous vocal fold medial surface. A sinusoidal shear force of 1g was applied at each point, and resultant displacement curve logged. Using a regression algorithm, the stiffness of the tissue was derived in units of grams force per millimeter displacement. Five readings were taken at each position. The results indicate that there is a linear increase in stiffness with respect to position, increasing as the measurements are taken further from the vocal fold. There is a gradual change in stiffness of the subglottal mucosa of a pig larynx.