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Objective: Treatment of vocal fold scarring remains challenging. We have previously reported the therapeutic effects of local injection of basic fibroblast growth factor (bFGF) in animal models and humans. A novel collagen/gelatin sponge (CGS) is capable of sustained release of bFGF, which compensates for its quick absorption in vivo, avoiding multiple injections. This study aimed to evaluate the biocompatibility and efficacy of the CGS in rat vocal fold fibroblasts prior to human trials. Methods: Fibroblasts extracted from Sprague-Dawley rat vocal folds were seeded onto a CGS and then cultivated with bFGF at concentrations of 0, 10, and 100 ng/mL. Vocal fold fibroblast morphology, adhesion, proliferation, and gene expression were measured under these 3-dimensional conditions. Results: Cells adhered to the CGS from day 1. Although no significant differences in cell morphology were detected, cell proliferation was accelerated by bFGF administration. Expression of endogenous bFGF and hepatocyte growth factor was significantly up-regulated at 10 ng/mL bFGF. The expression of procollagen I and procollagen III was significantly suppressed, whereas HAS-1 and HAS-2 were up-regulated at 10 and 100 ng/mL bFGF. Conclusion: The collagen/gelatin sponge is biocompatible with vocal fold fibroblasts and may be useful as a bFGF drug delivery system for the treatment of scarred vocal folds.

Objectives: Previous studies have indicated that although normal wound healing requires low levels of reactive oxygen species (ROS), excessive amounts of ROS impair wound healing. In injured vocal folds, this excess may result in dysphonia due to scarring that is difficult to treat. However, the expression of ROS during vocal fold wound healing has yet to be investigated. In this study, we assessed the expression and localization of ROS in injured vocal folds by immunohistochemical analysis. Methods: Vocal folds of Sprague-Dawley rats were unilaterally injured by stripping the mucosa under transoral endoscopy. The larynges were harvested at specific time points after injury and were immunohistochemically examined for 4-hydroxy-2-nonenal (4-HNE), an ROS marker, and for the presence of inflammatory cells. Results: We found that 4-HNE–immunopositive cells were significantly increased in the lamina propria of the injured vocal folds as compared to the normal vocal folds on postinjury days 1 and 3. More than half of the 4-HNE–immunopositive cells were also immunopositive for a macrophage- and granulocyte-specific antibody. Conclusions: This study suggests that a large amount of ROS is produced during early-phase wound healing, until postinjury day 3, and that this period may be crucial for regulating ROS levels. The results also suggest that inflammatory cells may contribute to ROS generation.

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow—derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from calf dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow—derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow—derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds.

Objectives The overarching goal of this line of research is to translate basic fibroblast growth factor (bFGF) treatment for vocal fold scarring into practical clinical use. In a previous canine investigation, we demonstrated that bFGF improves phonation threshold pressure, mucosal wave amplitude, and histologic measures in vocal folds treated after injury. In the present study, we studied the effects of bFGF on gene expression of the extracellular matrix and growth factors in rat vocal fold fibroblasts. Methods Fibroblasts harvested from the vocal folds of 5 rats were treated with 3 concentrations of bFGF (0, 10, and 100 ng/mL). The fibroblasts were collected at 24 hours and 72 hours after bFGF administration. Quantitative polymerase chain reaction was then used to investigate the gene expression of the investigated growth factors and extracellular matrices. Results The results revealed significantly down-regulated expression of procollagen I and significantly up-regulated expression of hyaluronic acid synthase (HAS) 2 and fibronectin in fibroblasts treated with bFGF. The administration of bFGF also resulted in the up-regulation of bFGF and hepatocyte growth factor (HGF). No changes in the expression of HAS-1, tropoelastin, or procollagen III were observed between the treatment and control conditions. Conclusions Treatment with bFGF induces the down-regulation of procollagen I and the up-regulation of HAS-2 in vocal fold fibroblast cell cultures. These gene expression alterations to key mediators of the wound healing process may translate into potential benefits in the remediation of vocal fold injury. The up-regulation of HGF, an antifibrotic effector molecule, may demonstrate additional benefits by optimizing the wound healing environment and by accelerating the wound repair cascade. These findings may provide fuel for additional discoveries into the development of growth factor therapy for the treatment of vocal fold scar.

Objectives: The management of vocal fold scarring and sulcus vocalis is challenging. These disorders are thought to be fibroplastic anomalies in the cover portion of the vocal fold that cause deterioration of the vibratory properties of the vocal fold mucosa. Histologic studies have revealed disorganization of extracellular matrix that needs to be addressed in the treatment of scarred vocal folds. Replacement of scar tissues with an appropriate implant may lead to regeneration of the vocal fold mucosa and its tissue properties. This retrospective case study examined the feasibility of using an atelocollagen sheet as a regenerative implant. Methods: Six patients with a post-cordectomy scar or sulcus vocalis underwent implantation of an atelocollagen sheet into the lamina propria of the vocal folds. The procedure consisted of elevation of a microflap, dissection and removal of scar tissue, implantation of the material, and wound closure. Vocal function was evaluated before and after surgery by stroboscopic examination and by aerodynamic and acoustic analyses. Results: The postoperative changes of aerodynamic and acoustic parameters varied among patients; however, gradual improvement was seen in most cases over a year. Stroboscopic findings also revealed gradual improvement of vibratory properties in most cases. Conclusions: Implantation of an atelocollagen sheet may have restorative effects on vocal fold scarring and sulcus vocalis in terms of tissue properties and function of the mucosa.

Objectives: Venous malformations are benign lesions with thin, fragile mucosa overlying a vascular stroma. Vascular anomalies often manifest as subglottic lesions in infants, but venous malformations in adults are rare in the pharyngolaryngeal region. The treatments include open and endoscopic surgery; intraoperative bleeding is often troublesome. Angiolytic lasers such as the potassium titanyl phosphate (KTP) laser enable photocoagulation for such hemorrhagic lesions without bleeding; we report findings from a series of adult patients. Methods: Seven adults were treated with a KTP laser set at a low power of 1.5 W in continuous mode. Photocoagulation was easily performed for shallow lesions; however, laser irradiation of bulky venous malformations resulted only in surface photocoagulation. In such cases, the crust remaining after photocoagulation was removed, and laser energy was repeatedly delivered until no remnant lesion was seen. An office procedure using flexible endoscopy was performed under topical anesthesia for 1 patient with a limited lesion. Results: The lesions were well controlled in all cases without major complications. A patient with a large obstructing lesion had a relapse. Because the recurrent lesion is small and the patient does not desire additional treatment at this time, she is being observed carefully. Conclusions: Photocoagulation using the KTP laser is a feasible and relatively safe treatment for pharyngolaryngeal venous malformations in adults.

Objectives: The regenerative processes of the vocal fold, or the existence of stem cells in the folds, are unknown. Side population (SP) cells are defined as cells that have the ability to exclude the DNA binding dye, Hoechst 33342. They are regarded as a cell population enriched with stem cells and can be isolated from non-SP cells by a fluorescence-activated cell sorter. This study was designed to determine whether SP cells exist in the human vocal fold, as a first step in elucidating the regenerative mechanisms of the vocal fold. Methods: Seven human excised larynges were used in this study. Two were used for fluorescence-activated cell sorter analysis, and 5 were subjected to immunohistochemical analysis with antibodies against an adenosine triphosphate binding cassette transporter family member, ABCG2, which is expressed in SP cells. Results: The number of SP cells in the human vocal fold was about 0.2% of the total number of cells. ABCG2-positive cells were identified in both the epithelium and subepithelial tissue throughout the entire vocal fold. Conclusions: This preliminary study demonstrated the existence of SP cells in the human vocal fold. Further studies are warranted to clarify how these cells work in the vocal fold, particularly in the regenerative process.

Objectives: Hypopharyngeal cancer (HPC) and cervical esophageal cancer (Ce) are aggressive tumors with a poor prognosis. Multiple lymph node metastases often occur in the upper mediastinum, as well as in the neck, and thus upper mediastinal dissection (MD) is crucial to improving the cure rate. However, excessive MD can increase postoperative morbidity and mortality, making it important to employ the proper technique and appropriate extent of dissection. In the present retrospective study we aimed to determine the proper extent of upper MD according to tumor site and stage. The benefit and risk of upper MD are also discussed. Methods: Chart review was completed for patients who underwent upper MD, including 64 patients with HPC, 21 patients with Ce, and 9 patients with Ce extending to involve the upper thoracic esophagus (Ce/Ut). The incidence and distribution of lymph node metastases in the upper mediastinum were assessed by postoperative histopathologic examination. Postoperative complications of upper MD, as well as the impact on survival and locoregional control, were also reviewed. Results: Upper mediastinal metastases were detected in 7.8% of HPC patients, 33.3% of Ce patients, and 55.6% of Ce/Ut patients. In HPC patients, mediastinal metastases were usually associated with T4 primary tumors (80%), whereas positive nodes in the upper mediastinum were detected regardless of T stage in both Ce and Ce/Ut. Only 1 Ce/Ut patient with a T4 tumor developed late nodal metastasis in the lower mediastinum. The 5-year disease-specific survival and locoregional control rates were 58.6% and 90.2% in HPC, 45.5% and 94.1% in Ce, and 38.9% and 77.7% in Ce/Ut, respectively. Rupture of the greater vessels after MD was observed in 5 cases (5.3%). Conclusions: The present results indicate excellent locoregional control rates following upper MD, while major complications such as arterial breakdown were rare. It is suggested that upper MD may be an essential and adequate procedure for patients with Ce or Ce/Ut tumors, and may also be required for cases of HPC with a T4 primary to improve locoregional control of the disease.

Objectives: Ectasias and varices of the vocal fold are microvascular lesions that are often due to chronic abuse of the voice, and are occasionally encountered in association with other disorders such as polyps, Reinke's edema, and hematoma. The KTP laser can be used for photocoagulation of small vascular lesions, because the laser beam is well absorbed by hemoglobin, and damage to the epithelium is minimal. The present pilot study examined how the KTP laser could be used for microvascular lesions and their associated lesions. Methods: Twelve patients who had undergone phonomicrosurgery were enrolled in the present study. The microvascular lesions were treated by photocoagulation with the laser set at a low power of 1.5 W in the continuous mode, while preserving the epithelium, and associated lesions were then treated by microdissection with cold instruments. The postoperative phonatory function was assessed by maximum phonation time, a perceptual test rating (GRBAS scale), and stroboscopy. Results: The procedures were completed successfully in all cases. An exceptional case of a small hemorrhagic polyp allowed treatment with the laser only. The postoperative stroboscopic findings, maximum phonation time, and perceptual test rating all showed significant improvement compared with the preoperative state. No adverse effects, such as scarring or reduction of the mucosal wave, were observed in the current series. Conclusions: KTP laser photocoagulation is a relatively simple and safe procedure for treating microvascular lesions of the vocal fold. It is not recommended for photocoagulation of hemorrhagic polyps or hematomas, because such lesions have little blood flow inside and thus photocoagulation is usually impossible or requires too much laser energy. However, photocoagulation of perimeter or feeding vessels of such disorders may facilitate the following procedure by avoiding unnecessary bleeding, as well as preventing recurrence of hemorrhagic lesions.

Vocal fold scarring remains a therapeutic challenge. Hepatocyte growth factor (HGF) has strong antifibrotic activity and has proved to have therapeutic potential in restoration of scar tissues such as liver cirrhosis and lung fibrosis. The present study aimed to clarify the effects of HGF injection into scarred vocal folds in a canine model. Canine vocal folds were stripped unilaterally and treated with intracordal injection of saline solution (sham group), HGF (HGF group), or HGF with cultured autologous normal vocal fold fibroblasts (Fb/HGF group) 1 month after injury. The larynges were harvested 6 months after the initial injury and then subjected to vibratory and histologic examination. The results of vibratory examinations in the excised larynx setup revealed that phonation threshold pressure significantly increased and vocal efficiency was significantly reduced in all treated groups as compared to normal data obtained from normal canine larynges. However, the HGF group presented much better results than both the sham and Fb/HGF groups in terms of mucosal wave amplitude and incidence of vocal fold bowing, glottal incompetence, and phase asymmetry. The histologic data indicated a significant increase of collagen in both the sham and Fb/HGF groups, while normal levels of collagen were found in the HGF group. Tissue contraction of the lamina propria was also observed in both the sham and Fb/HGF groups, but was barely detectable in the HGF group. Although the HGF-treated vocal folds appeared to require more driving forces for vibration, HGF might prevent excessive collagen deposition and tissue contraction and thus reduce the effects of scarring on the vibratory properties of the vocal folds. From these data it is concluded that HGF has considerable potential in the treatment of vocal fold scarring.

The aim of this study was to regenerate the injured vocal fold by means of selective cultured autologous mesenchymal stem cells (MSCs). Eight adult beagle dogs were used for this experiment. Selective incubation of MSCs from bone marrow was done. These MSCs were submitted to 3-dimensional incubation in 1% hydrochloric acid atelocollagen. Three-dimensional incubated MSCs were injected into the left vocal fold, and atelocollagen only was injected into the right vocal fold of the same dog as a control. Four days after injection, the posterior parts of the vocal folds were incised. The regeneration of the vocal fold was estimated by morphological and histologic evaluations. Our results showed that 3-dimensional incubated MSCs were useful in the regeneration of the injured vocal fold. This study shows that damaged tissues such as an injured vocal fold would be able to be regenerated by tissue engineering.

Vocal fold fibrotic scar is characterized by fibrosis of the lamina propria and epithelium, and is difficult to treat. Hepatocyte growth factor (HGF) has antifibrotic activity and has received attention as a possible therapeutic alternative to treat fibrosis. In this study, in order to clarify whether HGF can be involved in vocal fold scarring, we examined the existence of HGF and its receptor, c-Met, in rat vocal folds, and then the activity of HGF in rabbit injured vocal folds, using immunohistochemistry and enzyme-linked immunosorbent assay. We found HGF and c-Met on epithelial cells and gland cells of the rat vocal folds. On the injured vocal folds of rabbits, little HGF was observed immediately after injury, but prominent activity occurred simultaneously with reepithelialization of the vocal fold mucosa on days 10 to 15. The activity of HGF was observed on fibroblasts in the lamina propria, as well as the epithelium. It is suggested that HGF in the vocal folds is produced by the fibroblasts and delivered to the epithelium. The implication of these findings is that HGF is involved in wound healing of the vocal fold, and may provide an alternative approach in preventing and treating vocal fold scarring.

To attain posterior medialization by thyroplasty type I, we simulated the surgery using 3 cadaveric larynges. Two approaches were applied to adduct the arytenoid cartilage: one involved compression of the vocal process, and the other, compression of the muscular process inward. The inner perichondrium was incised to reach the arytenoid cartilage from the framework. To compress the vocal process, a large silicone plug was required. In this method, there was some difficulty in compressing the vocal process precisely, and there was the risk of postoperative extrusion of the large plug. In contrast, the muscular process was more superficial relative to the framework, and it could be precisely compressed, resulting in sufficient posterior glottal closure. The silicone plug required for this procedure has a shallow depth that may provide the advantage of preventing postoperative extrusion. In conclusion, the compression of the muscular process is preferable for safely achieving posterior glottal closure.

Imperfect glottal closure is usually the most important factor causing dysphonia in patients with bowing of the vocal folds. We have performed laryngeal framework surgery, which allows the medialization of the vocal folds from the outside without creating any scar tissue on them. Over the past 6 years, however, we encountered three cases with marked bowing of the vocal folds that could not be cured by laryngeal framework surgery alone. We used an open laryngeal procedure in these cases, even though such procedures had been considered contraindicated in the treatment of hoarseness. After performing a laryngofissure, we made a small pocket beneath the vocal fold mucosa at the anterior commissure. The superiorly based omohyoid muscle flap was then transposed into the mucosal pocket and sutured to the vocal process. This procedure should be considered an option in treating highly bowed vocal folds.

The effectiveness of surgical treatment for vocal fold atrophy of various causes was examined. Type I thyroplasty was performed on 31 patients with vocal fold atrophy, often bilaterally and occasionally combined with type III thyroplasty. Of these 31 patients, 3 patients underwent the operation twice, for a total of 34 operations. The surgery was found to be effective in improving the voice quality and the ease of phonation. When a scar or sulcus was present, the results were not as satisfactory. The intraoperative decision-making process as to which procedure to perform is extremely important for achieving an optimal voice, and should be based on the intraoperative voice quality, fiberoptic findings, and manual tests. In operations for vocal fold atrophy in which the vocal folds are mobile, a silicone shim should be firmly fixed to prevent migration. Overcorrection is generally recommended. No complications were encountered. It was often the increased ease of phonation rather than the improved voice quality that patients appreciated after surgery.