Abstract
Purpose
The purpose of this prospective study was to determine the effect of autologous transplantation of fascia into the vocal fold (ATFV) with controlled release of basic fibroblast growth factor (bFGF) on unilateral vocal fold paralysis (UVFP) in a rat model.
Materials and methods
Unilateral recurrent laryngeal nerve (RLN) section was performed on 15 rats. Ten rats received an autologous fascia implant and gelatin hydrogel with or without bFGF (1 μg) to their larynxes (fascia only, “fascia group”; bFGF + fascia, “fascia + bFGF group”), while the rest underwent RLN transection (“RLN section group”). Four months later, evaluation of the laryngeal glottal gap and histological analysis were performed.
Results
The glottal gap was significantly reduced in the fascia + bFGF group, and fat volume increased significantly relative to the RLN section. The volume of the remaining fascia in the bFGF + fascia group was significantly greater than that of the fascia group.
Conclusions
ATFV with controlled release of bFGF may compensate for diminished laryngeal volume in UVFP by reducing resorption of the implanted fascia and increasing fat volume. Our findings suggest that this modality may represent an attractive option for treating UVFP.
1
Introduction
In unilateral vocal fold paralysis, hoarseness and aspiration result from incomplete glottic closure. Repair of glottic incompetence may help avoid deterioration in conversation ability and reduce the risk of aspiration pneumonia.
Management of incomplete glottic closure includes laryngeal framework surgery, arytenoid adduction, and vocal fold injection . In addition, we have presented case histories outlining the success of autologous transplantation of fascia into the vocal fold (ATFV) to improve unilateral vocal fold paralysis . Our findings suggested that this technique could regenerate atrophied vocal fold tissue and produce adequate long-term results . Regeneration of damaged tissue can be achieved with a combination of scaffolds, cells, and regulatory factors under appropriate conditions . ATFV donates the scaffold and cells; however, the requisite growth factors and cells may not always migrate to the implanted area , and furthermore, the expression of growth factors in host tissue is unstable after implantation. Therefore, we hypothesized that ATFV combined with growth factor administration would improve vocal fold tissue regeneration.
Basic fibroblast growth factor (bFGF) is a strong promoter of angiogenesis that triggers proliferation of fibroblasts and mobilization of inflammatory cells from granulation tissue ; however, it has a short half-life. Tabata et al. presented a solution with controlled release of bFGF through an acidic gelatin hydrogel , an administration system that could be used in association with ATFV.
In this study, we examined histological and morphological changes in paralyzed vocal folds to evaluate the effect of ATFV with bFGF on improving glottic incompetence. A successful outcome would demonstrate that this method is a promising treatment for unilateral vocal fold paralysis.
2
Materials and methods
Male Sprague–Dawley rats (SD rats; age, 4–8 months; mean body weight, 588.3 ± 80.9 g) were used in this study.
This study was performed in accordance with the Kitasato University of Medicine Animal Care Manual. The animal use protocol was approved by the Ethics Committee of Kitasato University (Approval Number: 2009-1446, 2010-090, 2011-022).
2.1
Unilateral vocal fold paralysis model
SD rats underwent left recurrent laryngeal nerve (RLN) section to simulate vocal paralysis. First, the rats were anesthetized with pentobarbital solution (0.5 mg/kg) intraperitoneally after ether anesthesia and placed in a supine position. Second, a midline incision was made to the anterior neck, and the strap muscle was elevated to expose the omohyoid muscle above the trachea on the left side. After sectioning the omohyoid muscle from its attachment to the hyoid bone, the left RLN was identified, ligated to remove 3–5 mm, and sutured at both ends using 8.0 nylon.
2.2
Preparation of gelatin hydrogel sheets containing bFGF
Gelatin hydrogels were prepared from an aqueous solution of glutaraldehyde and gelatin, as previously reported . With the water content of the hydrogel sheet at 96 wt%, the duration of bFGF release for the present study was two weeks . The gelatin hydrogel was trimmed into 2 × 2 mm squares, which were then soaked in an aqueous solution containing 1 μg bFGF at room temperature three hours before use.
2.3
Experimental procedure for implantation
Autologous thoracolumbar fascia was harvested with the surrounding connective tissue attached. The fascia was squeezed tightly to dehydrate it and then shaped into a 2 × 2 mm square.
Kumai et al. reported a significant reduction in the area and fiber size of thyroarytenoid (TA) muscle two weeks after RLN section . Accordingly, implantation into the larynx was performed one month after RLN section, as histological alterations from laryngeal reorganization due to vocal fold paralysis should be recognizable at that point.
One group of rats that underwent RLN section received an implant with the fascia and gelatin hydrogel containing 1 μg bFGF (fascia + bFGF group, n = 5), another group received the fascia and gelatin hydrogel without bFGF (fascia group, n = 5), and a third group received no implant (RLN section group, n = 5). All animals were euthanized four months after RLN section.
2.4
Implantation of fascia and gelatin hydrogel with bFGF into the larynx
Nishiyama et al. reported that ATFV improved the voice quality of patients with unilateral vocal fold paralysis after surgery . However, it was difficult to adopt the same surgical methods in the rat larynx due to its small size. A procedure similar to type I thyroplasty was used instead to modify the approach .
A midline incision was made in the anterior neck of rats placed in the supine position. The strap muscle was then cleaved at its attachment to the hyoid bone, exposing thyroid cartilage and cricoid cartilage. The left cricothyroid muscle was incised at the inferior edge of the thyroid cartilage, and the thyroid lamina was sectioned vertically at the posterior middle third without injuring the pharyngeal mucosa. The fascia and hydrogel sheet were planted on the implantation bed, located on the lateral side of the TA muscle and lateral cricoarytenoid muscle. The surgical wound in the thyroid cartilage was then covered by the sutured strap muscle to protect the implant site ( Fig. 1 ). When necessary, a binocular loupe (Nihon Light, Japan) was used for the procedure.
2.5
Histological and morphological analysis
Four months after RLN section, a total laryngectomy was performed on each rat following ether anesthesia and an intracardiac injection of a lethal dose of pentobarbital solution. Each larynx was fixed with 10% formalin, dehydrated with graded concentrations of ethanol, and embedded in paraffin. Serial coronal paraffin sections (3 μm thick) were made at 100-μm intervals and stained with standard hematoxylin and eosin ( Fig. 2 ). The stained slides were viewed and photographed at low power with a digital camera (Canon, Japan) and light microscope (Nikon, Japan). A micrometer slide (Matsunami, Japan) was simultaneously photographed with each specimen for measurement standardization. The images for each slide were saved and constructed for the whole laryngeal image with Adobe Photoshop Elements (Adobe, USA). Two authors (H.N, K.N) blind to the experimental groups analyzed the slides to evaluate morphological changes in glottal gap area and histological changes in adipose tissue and implanted fascia.
2.6
Evaluation of glottal gap area
All laryngeal images were edited for use in Photoshop. Each image was divided in two down a middle line, with only the left, treated side used for analysis. The images were then superimposed at two data points, one at the intersection between the middle line and posterior surface of the laryngeal cavity and the other at the middle line. The glottal gap area of this superimposed image was then measured between the middle line and minimum laryngeal cavity line. The superimposed images were imported into Scion Image to measure the minimum area (a) and length from the anterior to posterior commissure at the middle line (L) (Scion, USA) ( Fig. 3 ). The determined glottal gap area was normalized to L, and the rate of glottal gap closure was calculated using the following formula: normalized glottal gap area (units) = a / L 2 × 100 units .
2.7
Evaluation of fascia and fat volume
All laryngeal images were imported into Scion Image for analysis. The volume of implanted fascia and fat tissue in each image was estimated using the following formula: V (mm 3 ) = area (sum of multiple sections) × 100 μm. The volume of fat in the untreated (U) and implanted (I) sides of the paraglottic space in the larynx was measured to determine the percentage of fat (I/U) for each animal. This ratio measures change in the fat volume of paralyzed vocal folds after surgery.
2.8
Statistical analysis
The present study evaluated a continuous response variable from independent control and experimental subjects, with one control per subject. In a previous study , the response within each subject group was normally distributed with standard deviation of 0.1. With the true difference in experimental and control means set at 0.2, we estimated that the sample size of five animals per group would be sufficient to reject the null hypothesis that the population means of the two groups are equal with probability (power) 0.8. The Type I error associated with the null hypothesis test was 0.05.
All data are presented as mean ± standard deviation. Differences in volume of implanted fascia remaining were compared between the fascia + bFGF group and fascia group with the Mann–Whitney U test. Glottal gap area and fat volume percentage were compared between groups using the Kruskal–Wallis rank test. Significant differences were analyzed with the Bonferroni–Dunn test for multiple comparisons. Stat View for Windows (Ver. 5.0, SAS Institute Inc.) was used for statistical analyses, and p < 0.05 was considered statistically significant.
2
Materials and methods
Male Sprague–Dawley rats (SD rats; age, 4–8 months; mean body weight, 588.3 ± 80.9 g) were used in this study.
This study was performed in accordance with the Kitasato University of Medicine Animal Care Manual. The animal use protocol was approved by the Ethics Committee of Kitasato University (Approval Number: 2009-1446, 2010-090, 2011-022).
2.1
Unilateral vocal fold paralysis model
SD rats underwent left recurrent laryngeal nerve (RLN) section to simulate vocal paralysis. First, the rats were anesthetized with pentobarbital solution (0.5 mg/kg) intraperitoneally after ether anesthesia and placed in a supine position. Second, a midline incision was made to the anterior neck, and the strap muscle was elevated to expose the omohyoid muscle above the trachea on the left side. After sectioning the omohyoid muscle from its attachment to the hyoid bone, the left RLN was identified, ligated to remove 3–5 mm, and sutured at both ends using 8.0 nylon.
2.2
Preparation of gelatin hydrogel sheets containing bFGF
Gelatin hydrogels were prepared from an aqueous solution of glutaraldehyde and gelatin, as previously reported . With the water content of the hydrogel sheet at 96 wt%, the duration of bFGF release for the present study was two weeks . The gelatin hydrogel was trimmed into 2 × 2 mm squares, which were then soaked in an aqueous solution containing 1 μg bFGF at room temperature three hours before use.
2.3
Experimental procedure for implantation
Autologous thoracolumbar fascia was harvested with the surrounding connective tissue attached. The fascia was squeezed tightly to dehydrate it and then shaped into a 2 × 2 mm square.
Kumai et al. reported a significant reduction in the area and fiber size of thyroarytenoid (TA) muscle two weeks after RLN section . Accordingly, implantation into the larynx was performed one month after RLN section, as histological alterations from laryngeal reorganization due to vocal fold paralysis should be recognizable at that point.
One group of rats that underwent RLN section received an implant with the fascia and gelatin hydrogel containing 1 μg bFGF (fascia + bFGF group, n = 5), another group received the fascia and gelatin hydrogel without bFGF (fascia group, n = 5), and a third group received no implant (RLN section group, n = 5). All animals were euthanized four months after RLN section.
2.4
Implantation of fascia and gelatin hydrogel with bFGF into the larynx
Nishiyama et al. reported that ATFV improved the voice quality of patients with unilateral vocal fold paralysis after surgery . However, it was difficult to adopt the same surgical methods in the rat larynx due to its small size. A procedure similar to type I thyroplasty was used instead to modify the approach .
A midline incision was made in the anterior neck of rats placed in the supine position. The strap muscle was then cleaved at its attachment to the hyoid bone, exposing thyroid cartilage and cricoid cartilage. The left cricothyroid muscle was incised at the inferior edge of the thyroid cartilage, and the thyroid lamina was sectioned vertically at the posterior middle third without injuring the pharyngeal mucosa. The fascia and hydrogel sheet were planted on the implantation bed, located on the lateral side of the TA muscle and lateral cricoarytenoid muscle. The surgical wound in the thyroid cartilage was then covered by the sutured strap muscle to protect the implant site ( Fig. 1 ). When necessary, a binocular loupe (Nihon Light, Japan) was used for the procedure.
