Atsushi Suehiro

Chronic vocal fold scar restoration with hepatocyte growth factor hydrogel

Therapeutic challenges exist in the management of vocal fold scarring. We have previously demonstrated the therapeutic potential of hepatocyte growth factor (HGF) in the management of acute phase vocal fold scarring using a novel hydrogel‐based HGF drug delivery system (DDS). However, the effect of HGF on matured vocal fold scarring remains unclear. The current study aims to investigate the effect of HGF‐DDS on chronic vocal fold scarring using a canine model.

Drug delivery system of hepatocyte growth factor for the treatment of vocal fold scarring in a canine model.

Objectives: Vocal fold scarring remains a therapeutic challenge. Previous studies have indicated that hepatocyte growth factor (HGF), a strong antifibrotic element, has therapeutic potential for restoring scarred vocal folds. To enhance the effect of HGF in vivo, we developed a novel drug delivery system (DDS) in which HGF is embedded in gelatin hydrogel and continuously released over a period of 2 weeks. In the present study we investigated the therapeutic efficacy of the HGF DDS on vocal fold scarring by using a canine model. Methods: The vocal folds of 8 beagles were unilaterally scarred by stripping the entire layer of the lamina propria. The contralateral vocal folds were kept intact as normal controls. One month after the procedure, hydrogels (0.5 mL) containing 1 μg of HGF were injected into the scarred vocal folds of 4 dogs (HGF-treated group), whereas hydrogels containing saline solution were injected in the other 4 dogs (sham group). Histologic and vibratory examinations were completed for each group 6 months after the initial surgery. Results: The excised larynx experiments showed significantly better vibration in terms of mucosal wave amplitude and glottal closure in the HGF-treated group compared to the sham group. Histologic evaluation of the vocal folds indicated remarkable reduction in collagen deposition and tissue contraction, with favorable restoration of hyaluronic acid and elastin in the HGF-treated group. Conclusions: The present findings suggest that the novel HGF DDS may provide favorable effects in restoring the vibratory properties of scarred vocal folds.

Regeneration of aged vocal fold: first human case treated with fibroblast growth factor.

BACKGROUND/OBJECTIVES Aged vocal folds are characterized by atrophy of the mucosa, which caused dysphonia and is difficult to treat. We have revealed a therapeutic potential of basic fibroblast growth factor (bFGF) for tissue regeneration of the aged vocal fold. We report here the first human case that has been treated with bFGF. STUDY DESIGN Institutional review board-approved clinical human trial. METHODS A 63-year-old male with atrophied vocal folds was treated by local injection of 10 mug of bFGF into the left vocal fold under topical anesthesia. The effects of the injection were examined after 1 to 3 months by videostroboscopy, acoustic, and aerodynamic measurements. RESULTS The atrophy of the vocal fold was improved at 1 week after the injection, and glottic gap disappeared. Aerodynamic and acoustic parameters also showed remarkable improvement. These positive effects were maintained up to 3 months. CONCLUSIONS The first case with aged vocal folds treated with bFGF administration was presented. The results are encouraging, suggesting therapeutic effects of bFGF for atrophied vocal folds in human.

Treatment of acute vocal fold scar with local injection of basic fibroblast growth factor: a canine study

Abstract Conclusions: Results of the current study revealed improved phonation threshold pressure (PTP), normalized mucosal wave amplitude (NMWA), and less contraction of the lamina propria in injured larynges treated with basic fibroblast growth factor (bFGF). Objectives: We investigated the effects of local injection of bFGF for treatment of acute vocal fold injury in a canine model. Methods: Vocal folds of eight beagles were unilaterally injured by removal of the mucosa under direct laryngoscopy. Four beagles received local injections of bFGF delivered to the scarred vocal fold at 1 month after injury. The remaining four beagles received local injections of saline and served as a sham-treatment group. Larynges were harvested 5 months after treatment and excised larynx experiments were performed to measure PTP, NMWA, and normalized glottal gap (NGG). Histologic staining was performed to evaluate structural changes of the extracellular matrix. Results: Excised larynx measurements revealed significantly lower PTP and increased NMWA in bFGF-treated vocal fold. Elastica Van Gieson staining revealed less contraction of the bFGF-treated vocal fold. Histologic measurements revealed that the thickness of the lamina propria was significantly greater in the bFGF-treated vocal fold. Alcian blue staining revealed improved restoration of hyaluronic acid in the bFGF-treated vocal fold.

Endoscopic KTP Laser Photocoagulation Therapy for Pharyngolaryngeal Venous Malformations in Adults

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.

Raised intensity phonation compromises vocal fold epithelial barrier integrity.

We investigated the hypothesis that 30 minutes of raised intensity phonation alters transcript levels of vocal fold intercellular tight junction proteins and disrupts the vocal fold epithelial barrier.

Effects of basic fibroblast growth factor on rat vocal fold fibroblasts.

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.

Side population cells in the human vocal fold.

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.

Atelocollagen Sponge as a Stem Cell Implantation Scaffold for the Treatment of Scarred Vocal Folds

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.

Effect of exogenous hepatocyte growth factor on vocal fold fibroblasts.

Objectives We have previously demonstrated the therapeutic potential of hepatocyte growth factor (HGF) in the treatment of vocal fold scarring, although how exogenous HGF affects gene expression of endogenous HGF or extracellular matrix components in the vocal fold fibroblasts remains unclear. In this in vitro study, we aimed to clarify this aspect in order to better understand the effects of HGF on the vocal folds. Methods Fibroblasts were obtained from the lamina propria of the vocal folds of 5 Sprague-Dawley rats and were cultured with HGF at concentrations of 100, 10, 1, and 0 ng/mL. The cells were collected on days 1, 3, and 7, and the expression of endogenous HGF, its receptor c-Met, transforming growth factor-β1 (TGF-β1), procollagen types I and III, and hyaluronic acid synthase (HAS)-l and HAS-2 messenger RNAs (mRNAs) was examined by real-time reverse transcription-polymerase chain reaction. Results The expression of endogenous HGF and HAS-1 mRNAs increased significantly when exogenous HGF was administered at a concentration of 1 ng/mL. On day 1, the expression of TGF-β1 and HAS-2 mRNAs increased significantly in response to 1 ng/mL HGF. Conclusions Exogenous HGF triggered the up-regulation of endogenous HGF, TGF-β1, HAS-1, and HAS-2 mRNAs in vocal fold fibroblasts.