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Objectives: The aims of this study are to investigate the efficiency of a tissue engineering approach to partial tracheal reconstruction and to improve epithelialization of the reconstructed trachea. The trachea must be resected in some cases of cancer or trauma. Various restructuring techniques are used, with no consensus on the best approach. Two problems that arise when treating tracheal defects by conventional techniques are an inability to regenerate ciliated epithelium at the reconstructed site and having to perform multiple procedures to achieve the desired repair. This study is designed to address these problems. Study Design: Preliminary, an animal experiment. Methods: Surgery was performed on five adult beagles under anesthesia. After the making of a longitudinal cervical skin incision, the trachea was exposed and a circular defect created. A polypropylene and collagen scaffold preclotted with peripheral blood was inserted to the defect site. Postoperatively, the site was evaluated fiberscopically, histologically, and radiographically. Results: All dogs did well postoperatively. Fiberscopic examination showed that the implanted scaffolds were completely covered with regenerated mucosa with capillaries in all cases. Histologic data showed ciliated epithelium regenerated at the operated site from 1 month postoperatively. Newly formed cartilage was detected in the specimens from 8 to 12 postoperative months. Computed tomography images revealed the fine luminal contour of the regenerated site. Conclusions: Good epithelial regeneration was observed after repair of a round tracheal resection using a simple tissue engineering technique, making the technique a good substitute for conventional approaches to tracheal reconstruction in patients with cancer or trauma. [ABSTRACT FROM AUTHOR]

Objectives/hypothesis: Vocal fold scar remains a therapeutic challenge. Mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. Nevertheless, few in vivo studies have directly compared various sources of MSCs. The aim of this study was to investigate the therapeutic potential of adipose-derived stromal cells (ASCs) in comparison with bone marrow-derived stromal cells (BMSCs) for vocal fold regeneration., Study Design: Prospective animal experiments with controls., Methods: Two months after stripping of the lamina propria, 18 beagles were divided into four implantation groups: atelocollagen alone (collagen group), atelocollagen with BMSCs (BMSC-collagen), atelocollagen with ASCs (ASC-collagen), or a sham-treated group. One or 6 months after implantation, vibratory and histological examinations were performed., Results: Mucosal vibration was significantly improved in both of the MSC-implanted groups compared with the sham-treated group, whereas only the ASC-collagen group showed a significantly smaller glottal gap than the collagen group. Moreover, in the ASC-collagen group, a significant reduction of collagen density was observed compared to the sham-treated group, and there was a trend for better restoration of hyaluronic acid (HA). Implanted MSCs were detected 1 month postimplantation; however, none survived 6 months postimplantation., Conclusions: Although implantation of an atelocollagen sponge and ASCs into vocal fold scars induced vibratory recovery comparable to that of BMSCs, ASCs might have more potential in terms of restoration of HA and suppression of excessive collagen deposition., Level of Evidence: NA Laryngoscope, 126:1143-1150, 2016., (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Stem cells are known to proliferate at a slow rate in adult organs, and thus slow-cycling cells exhibiting pluripotency are considered tissue-specific stem cells in some organs. Slow-cycling cells in the vocal fold (VF) have not been well documented. Here we sought to clarify the distributions and characteristics of slow-cycling cells in rat VFs., Methods: We applied double-labeling technique to detect the distribution of slow-cycling cells. We injected the exogenous proliferation marker 5-bromo-2'-deoxyuridine (BrdU) into Sprague-Dawley rats. After a chasing period, VFs were immunostained with antibodies to BrdU and the second endogenous proliferation marker, Ki-67. BrdU (+) Ki-67(+) cells were regarded as slow-cycling cells and counted by VF regions. To reveal slow-cycling cells' characteristic, their immunophenotypes were histologically investigated and their kinetics in injured VFs were evaluated., Results: Most slow-cycling cells were detected in the basal layer of the epithelium. Slow-cycling cells in the epithelium displayed a low positive ratio of E-cadherin and CK5 and a high positive ratio of vimentin and CD31 as compared with the other epithelial cells. The expression of S100A4 was low in slow-cycling cells of the lamina propria and the macula flava. FGFR1, HAS1, HAS2, and HAS3 were not detected in the slow-cycling cells. A time-dependent reduction of slow-cycling cells was observed in injured VFs., Conclusion: Most slow-cycling cells resided in the epithelium, exhibiting various phenotypes in a relatively undifferentiated condition, and they are suspected to contribute to the tissue repair of the injured VFs., Level of Evidence: N/A., (© 2016 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Vocal fold scarring presents therapeutic challenges. Recently, cell therapy with mesenchymal stromal cells has become a promising approach. The aim of this study was to compare the therapeutic potential of adipose-derived stem cells (ASC) with bone marrow-derived stem cells (BMSC) for vocal fold regeneration., Study Design: Prospective animal experiments with controls., Methods: The vocal folds of Sprague-Dawley rats were unilaterally injured. Two months after injury, rats were treated with a local injection of ASC (ASC group), BMSC (BMSC group), or saline (sham-treated group). The GFP-labeled ASC and BMSC were extracted from CAG-EGFP rats. Larynges were harvested for histological and immunohistochemical examinations 1 and 3 months posttransplantation and for quantitative real-time polymerase chain reaction (PCR) 1 month posttransplantation., Results: After 1 month, no surviving cells from the transplant were detected. Histological examination showed significantly increased hyaluronic acid (HA) and decreased dense collagen deposition in both ASC and BMSC groups compared to shams 1 and 3 months after treatment. Real-time PCR revealed that hyaluronan synthase 1 (Has1) and Has2 were upregulated in only the ASC group compared with the sham-treated group. Fibroblast growth factor 2 (basic) (Fgf2), hepatocyte growth factor (Hgf) and Has3 were upregulated in both cell transplantation groups. ASC seemed to upregulate Hgf more than did BMSC., Conclusions: The regenerative effects of ASC and BMSC transplantation were found to be similar for the restoration. It is suggested that ASC might have more potential because of better recovery of HA, a superior antifibrotic effect, and the upregulation of Hgf., Level of Evidence: N/A., (© 2014 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Reactive oxygen species (ROS) are associated with aging. Astaxanthin (AST) is a strong antioxidant and has been reported to prevent various ROS-induced diseases. In the current study, we investigated the effect of AST on age-associated histological and mRNA changes of vocal folds., Study Design: Prospective animal experiment with control., Methods: Six-month-old Sprague-Dawley rats were fed on a normal powder diet with 0.01% (w/w) AST (aged AST-treated group) or without AST (aged sham-treated group). After 12 months of feeding, the larynges were harvested for histology, immunohistochemical detection of 4-hydroxy-2-nonenal (4-HNE), and quantitative real-time polymerase chain reaction for basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF). Thirteen-week-old rats were used as a young control group (young group)., Results: The expression of 4-HNE, an oxidative stress marker, significantly increased in the two aged groups compared with the young group. Histological examination showed that the deposition of hyaluronic acid in the lamina propria (LP) was significantly reduced in the aged sham-treated group compared with the young group, but no significant difference was observed between the aged AST-treated group and the young group. There were no significant differences in the mRNA expression of bFGF and HGF between the aged AST-treated group and the young group, although the expression of these genes was significantly reduced in the aged sham-treated group as compared with the young group., Conclusions: These results suggest that AST has the potential to attenuate age-associated changes of vocal folds., (© 2014 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Previous animal studies demonstrated that hepatocyte growth factor (HGF) has the potential to regenerate scarred vocal folds. In addition, HGF is now produced under a good manufacturing practice (GMP) procedure. Therefore, human clinical trials of HGF are warranted in patients with vocal fold scarring. In the current study, we investigated the pharmacokinetics and the local tissue responses of HGF administered to rat vocal folds., Study Design: Prospective animal experiment., Methods: Five μg of recombinant human HGF was administered to the vocal folds of Sprague-Dawley rats (n = 60) using a microsyringe. The concentration of HGF in larynges and blood was investigated by enzyme-linked immunosorbent assay. To evaluate the local tissue responses caused by HGF administration, endoscopic and histological examinations were performed., Results: HGF concentration in the larynges was 50.1 μg/g tissue 5 minutes after administration. The concentration decreased rapidly to 1.71 μg/g tissue at 12 hours after administration and to 0.29 ng/g tissue at 24 hours after administration. Seven days after administration, HGF concentration was minimal in one-half of the cases and was not detected in the other cases. Transmission of HGF to blood was detected in two of six cases at 5 minutes after administration, but was no longer detected 12 hours later. Endoscopic and histological examinations revealed no edema or erythema of the vocal folds in any of the cases., Conclusions: The current results contribute to the safety and pharmacokinetic management of future clinical trials using HGF administered to vocal folds., (© 2014 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Our previous studies demonstrated a regenerative effect of recombinant human hepatocyte growth factor (HGF) on vocal fold scarring using full-length HGF. However, clinical application has not yet been achieved because of the lack of a good manufacturing practice (GMP) for full-length HGF. Another natural form of human HGF, 5 amino acid-deleted type HGF (dHGF), has been newly produced under a GMP procedure. In the current study, we investigated the effect of dHGF in comparison with full-length HGF for the treatment of vocal fold scars using a canine model., Study Design: Prospective animal experiment., Methods: The vocal folds of nine beagles were unilaterally injured. Four weeks after injury, the vocal folds were treated with an intracordal injection of full-length HGF (full HGF group), dHGF (dHGF group), or phosphate-buffered saline (sham group). Vibratory and histological examinations were performed for each group 6 months after injury., Results: Vibratory examinations demonstrated significantly lower phonation threshold pressure and a higher ratio of normalized mucosal wave amplitude in both the full HGF and dHGF groups as compared to the sham group. Histological examination showed restoration of hyaluronic acid in both the full HGF and dHGF groups as compared to the sham group. No significant differences were observed for each parameter between the full HGF group and the dHGF group., Conclusions: dHGF showed the same potential for regenerative effects on vocal fold scars as full-length HGF. dHGF should be applicable for human clinical trials in patients with vocal fold scars., (© 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Our previous study demonstrated that a large amount of reactive oxygen species (ROS) is produced during the early phase of vocal fold wound healing. In the current study, we investigated the effect of astaxanthin, which is a strong antioxidant, on the regulation of oxidative stress and scarring during vocal fold wound healing., Study Design: Prospective animal experiment with control., Methods: Sprague-Dawley rats were dosed with astaxanthin (Ast-treated group, 100 mg/kg/day) or olive oil (sham-treated group) by oral gavage daily from preinjury day 1 to postinjury day 4. After vocal folds were injured under the endoscope, larynges were harvested for histological and immunohistochemical examinations on postinjury days 1, 3, 5, and 56, and quantitative real time polymerase chain reaction (PCR) on postinjury days 1 and 3., Results: The expression of 4-hydroxy-2-nonenal, which is an oxidative stress marker, was reduced significantly in the lamina propria of the Ast-treated group as compared to the sham-treated group. Histological examination showed significantly less tissue contraction with favorable deposition of hyaluronic acid in the lamina propria of the Ast-treated group compared to the sham-treated group. Real time PCR revealed significantly upregulated mRNA expression of basic fibroblast growth factor on postinjury day 1 and procollagen type I in the Ast-treated group compared to the sham-treated group., Conclusions: These findings suggest that astaxanthin has the potential to prevent vocal fold scarring by regulating oxidative stress during the early phase of vocal fold wound healing., (© 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Laryngeal venous malformation is a comparatively rare condition in adults. It presents as a livid or bluish mass that may cause bleeding, hoarseness, stridor, and even difficulty in breathing. So far, treatment has largely consisted of laser therapy and surgical removal. Five cases of adult laryngeal venous malformations that were treated by sclerotherapy (local injection of monoethanolamine oleate solution) are reported. All lesions disappeared after one or two treatment sessions, and two of the five cases that presented recently were cured in the outpatient clinic setting with no sequelae. Sclerotherapy with monoethanolamine oleate is a safe, easy, and effective treatment for laryngeal venous malformations., (Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: To investigate the functional efficiency of skeletal muscles regenerated by transplantation of bone marrow-derived stromal cells (BSCs) or induced-muscle progenitor cells (IMCs) as assessed in the canine posterior cricoarytenoid (PCA) muscle injury model., Study Design: Prospective animal experiment with control., Methods: We performed BSC/IMC transplantation into injured canine PCA muscles. We investigated the capability of auto- and allo-BSC/IMC transplantation using a gelatin sponge scaffold to promote functional regeneration of PCA muscles. Transplantation was assessed by fiberscopic analysis of vocal fold movement. We also examined the histologic changes of the transplanted regions. As a control, a gelatin sponge scaffold without additional cells was transplanted into the injured area., Results: Auto-BSC/IMC transplantation effectively restored vocal fold movement, whereas scaffold alone or allo-BSC/IMC transplantation did not. Histologic examination revealed that (in cases of good recovery) muscle regeneration occurred in the area of cell transplantation, and scar formation without muscle regeneration was observed under control conditions. The dogs with autologous transplantation of BSC had faster functional recovery than did dogs treated with autologous transplantation of IMC., Conclusions: Functional efficiency was shown in skeletal muscles regenerated using BSCs and IMPs. Motor function recovery was observed using autologous transplantation of BSCs and IMCs. Minimal functional recovery was observed using allogeneic transplantation of these cells., (Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Vocal fold scar and sulcus are still challenges. Basic fibroblast growth factor (bFGF) has proven to be effective to resolve scar tissue in animal models. This study reports the efficacy of regenerative treatments using bFGF on vocal fold scar and sulcus in human cases., Study Design: Retrospective chart review., Methods: Fifteen cases (7 scar; 8 sulcus) were treated by either local injection of bFGF (n = 6) or regenerative surgery using bFGF (n = 9). Injection regimen was to locally apply 10 micrograms of bFGF in 0.5 mL saline into each vocal fold under topical anesthesia repeatedly (4 times with intervals of 1 week between each injection). The regenerative surgical procedure consisted of the dissection of scar tissue and the implant of gelatin sponge with bFGF. Follow-up periods ranged from 6 months to 24 months., Results: Maximum Phonation Time (MPT); Voice Handicap Index (VHI)-10; and Grade, Roughness, Breathiness, Asthenia, Strain (GRBAS) scale were assessed in both groups. The injection group showed significant improvement on VHI-10 and GRBAS. The regenerative surgery group showed significant improvement in all parameters. Jitter and shimmer were evaluated in the surgery group, and the results indicated improvement in six and five cases of nine cases, respectively. No major adverse effects were observed in both treatment groups., Conclusions: Regenerative treatments using bFGF has shown to be effective for improvement of vocal function in scar and sulcus., (Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Most cases of chronic otitis media (OMC) are associated with poor development of the mastoid air cells (MACs) and poor Eustachian tube (ET) function. We have previously reported that MAC regeneration can effectively eliminate intractable OMC. In this study, we assessed the ability of regenerated MACs to restore normal gas exchange function and contribute to improved ET function., Study Design: Clinical trial with control., Setting: General hospitals., Materials and Methods: Seventy-six patients with OMC, including cholesteatoma and adhesive otitis media, received tympanoplasty and MAC regeneration therapy. At the first-stage of tympanoplasty, artificial pneumatic bones and/or autologous bone fragments were implanted into the opened mastoid cavity. At the 2nd-stage operation, a nitrous oxide (N2O) gas study was performed in 10 patients to measure middle ear pressure (MEP). For the control group, MEP was measured in five patients with good MAC development during cochlear implantation or facial nerve decompression. ET function was measured twice in each patient, once before the 1st operation and 6 months after the second operation., Results: At the 2nd-stage operation, in all cases with regenerated MACs and in the normal control group, MEP changed after administration of N2O. In contrast, no change in MEP was observed in cases with unregenerated MACs. In 70% (n = 37/53) of the regenerated MAC group, ET function was improved, whereas improvement of ET function was observed in only 13% (n = 3/23) of the unregenerated MAC group., Conclusions: Tissue-engineered regeneration of MACs improves ET function and gas exchange in the middle ear., (Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives/hypothesis: Vocal fold injury can cause intractable scarring resulting in dysphonia. Mesenchymal stem cells (MSCs) have great therapeutic potential in wound healing. They continuously circulate in the peripheral blood and migrate into wound sites where they induce regenerative effects. However, their roles in vocal fold wound healing are poorly understood because few MSCs exist in the peripheral blood and there is no specific marker to identify them. The present study evaluates how intravenously injected MSCs affect vocal fold wound healing using Green Fluorescent Protein (GFP) -labeled MSCs., Study Design: Prospective study using animal model., Methods: GFP-labeled MSCs were obtained from femurs of GFP transgenic Sprague-Dawley rats and incubated in culture. Sprague-Dawley rats underwent intravenous injection of GFP-labeled MSCs (1.0 × 10(6) cells) immediately after vocal fold injury. Histological examination was performed., Results: Injected MSCs were distributed throughout the vocal fold wound site from day 1 up to day 56. These vocal folds showed increased hepatocyte growth factor (HGF)-positive cells within the wound and improved wound healing compared with sham-treated folds., Conclusion: Circulating MSCs can migrate to vocal fold wound sites and upregulate the expression of HGF during wound healing; thus, they are considered to play a significant role in wound healing within the vocal folds., (Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives/hypothesis: Vocal fold scarring poses a therapeutic challenge. It causes hoarseness and decreases the quality of life. Transforming growth factor β3 (TGFβ3) is highly expressed in fetal wounds that heal without scarring, and administration of TGFβ3 has been reported to prevent scarring of the skin and the buccal mucosa. Thus TGFβ3 is considered to be a key molecule in scar-free healing. This study aimed to examine the ability of TGFβ3 to prevent vocal fold scarring, with particular attention paid to the distribution of extracellular matrices and functional outcomes., Study Design: Prospective study using an animal model., Methods: Ten beagles were used in this study; 500 μL of TGFβ3 (0.5 μg/mL: 5 beagles) or saline (5 beagles) was injected into the vocal fold lamina propria. Fifteen minutes after injection, vocal folds were injured by stripping off the entire layer of the lamina propria. Six months after surgery, animals were euthanized and the larynges were harvested. Vibratory and histologic examinations were performed., Results: The administration of TGFβ3 suppressed granulation-tissue formation and scarring. TGFβ3-treated vocal folds showed significantly better vibratory properties, resembling normal vocal folds. Histologic analysis revealed favorable restoration of elastin and hyaluronic acid in the lamina propria. The distribution of collagen was well organized, and collagen deposition was less dense in TGFβ3-treated vocal folds compared to sham-treated vocal folds., Conclusions: Administration of TGFβ3 before injury significantly suppressed scar formation and induced favorable restoration of extracellular matrices in the vocal fold lamina propria, resulting in much improved phonatory function., (Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives/hypothesis: Aged vocal folds are characterized by atrophy of the mucosa, which causes mucosal wave deficiency and glottal insufficiency. This clinical trial examined the regenerative effects and safety considerations of basic fibroblast growth factor (bFGF) on restoration of aged vocal folds., Study Design: Institutional review board-approved human clinical trial., Methods: Ten patients (6 men and 4 women; mean age, 70.1 years) were recruited in the trial. Ten micrograms of bFGF was injected into each treated vocal fold. Injection was performed unilaterally or bilaterally according to each patient's need and repeated up to seven times if necessary. Patient follow-up continued for at least 6 months. The effectiveness of the treatment was assessed by stroboscopic, acoustic, and aerodynamic measurements., Results: All patients showed improvement of voice. Significant improvements in maximum phonation time, mean flow rate, jitter, shimmer, and noise-to-harmonic ratio lasted for at least 1 year. No allergic or long-term adverse effects were noted., Conclusions: This clinical trial suggests that bFGF may be effective and safe as a regenerative agent for aged vocal folds., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives: Radiotherapy is one of the most effective treatments for head and neck cancer. However, the development of dry mouth syndrome is an unavoidable side effect because, in addition to the tumor, the normal salivary glands are included in the irradiation field. Previously, we investigated the protective efficacy of basic fibroblast growth factor (bFGF) in radiation-damaged salivary glands. In this study, we investigated the efficacy of adipose-derived stromal cell (ADSC) transplantation for the regeneration of radiation damaged salivary glands., Study Design: Prospective animal experiment with control., Methods: ADSCs (500,000 cells isolated from green fluorescent protein mice) were transplanted into submandibular glands of C57BL/6 mice 10 weeks after 10 Gy irradiation (ADSC group). Phosphate-buffered saline was administered to mice as a control (sham group). Submandibular gland morphology and saliva flow rate were assessed just prior to transplantation and at 5 and 10 weeks posttransplantation. Quantitative RT-PCR was performed to examine the response of irradiated submandibular glands to ADSC transplantation., Results: Saliva flow rate in the ADSC group was improved compared to the sham group. Although acinar cell damage was detected in both groups, the proliferation of blood vessels and other paracrine effects were observed only in the ADSC group. Additionally, the differentiation of ADSCs into blood endothelial cells and ductal cells was also observed., Conclusions: Our study indicates that ADSCs have the potential to restore salivary gland function after irradiation. The restoration of blood flow within submandibular gland tissue may explain the beneficial effects of ADSCs., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives/hypothesis: Radiotherapy is one of the most effective treatments for head and neck cancer. However, in addition to the target tumor, normal salivary glands are also included in the irradiation field. This unavoidably results in dry mouth syndrome as a side effect. In this study, the protective efficacy of basic fibroblast growth factor (bFGF) was investigated in radiation-damaged salivary glands., Study Design: Prospective animal experiment with control., Methods: Nine-week-old female C57BL/6 mice were divided into three groups. All mice in two of the three groups were irradiated (10 Gy) at the same time. In the bFGF-treated group, bFGF was administered to the submandibular glands for 3 consecutive days after neck irradiation. Mice in the untreated control group were administered distilled water. Mice in the third group were not irradiated and did not receive any additional treatments. Saliva flow rate and submandibular gland morphology were assessed, and the apoptotic response of irradiated submandibular glands was also evaluated., Results: Administration of bFGF improved hyposalivation 8 weeks after irradiation, and histologic analysis revealed that bFGF-treated glands contained more acinar cells compared to untreated glands. The apoptotic response to irradiation, examined 1 and 2 days after irradiation, was reduced, and quantitative real-time polymerase chain reaction revealed a paracrine effect for bFGF in the glands that received bFGF treatment., Conclusions: Our study indicates that bFGF prevents salivary gland dysfunction after irradiation. The protective benefits of bFGF may be attributed to the inhibition of radiation-induced apoptosis as well as the paracrine effect it has in these tissues., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives/hypothesis: Despite big advances in understanding the mechanisms of wound healing in vocal fold injury, it still remains unclear which are the decisive factors that lead to a complete restoration or to scarring. Among several other factors, stem cells are believed to play an important role in vocal fold restoration. Side population (SP) cells are considered to contain high numbers of stem cells and have gained great interest in the tissue engineering community. The aim of the following study was to investigate the recruitment pattern of SP cells in a rat vocal fold injury model., Study Design: Experimental animal study., Methods: Unilateral vocal fold scarring was performed in Sprague Dawley rats. Larynges were harvested 1, 3, 5, 7, 14, 21, and 35 days after initial injury and were examined immunohistochemically for the presence of SP cells. This was done in coronal sections of the posterior and anterior macula flava as well as in the midportion of the vocal fold investigating the lamina propria., Results: The number of SP cells peaked significantly after 7 days in the midportion of injured vocal folds, with a return to pre-injury levels after 14 days. No increase was detected throughout the observed time in the contralateral side. The number of SP cells increased slightly but not significantly in both anterior and posterior macula flava., Conclusions: Our findings suggest that SP cells may play an important role in early vocal fold wound healing and may serve as a possible therapeutic target., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives/hypothesis: In the treatment of tumorous diseases, scarring often forms after resection or irradiation. Scarring of the buccal mucosa causes difficulty in opening the mouth and mastication, decreasing quality of life. Transforming growth factor (TGF) β3 is an isoform of TGFβ1 that is known to accelerate scarring, although it has different effects on wound healing. TGFβ3 administration into wounds has been associated with improvement in the quality of healing skin in vivo. TGFβ3 is also considered to be an important anti-scarring factor in buccal mucosa. The present study aimed to examine whether TGFβ3 is effective for prevention and treatment of buccal mucosa scarring., Study Design: Prospective study using an animal model., Methods: Thirty Sprague-Dawley rats were involved in this study. We injected 0.5 mL of TGFβ3 (0.005 μg/mL, 0.05 μg/mL, 0.5 μg/mL, 5 μg/mL) or saline was injected into the buccal submucosa. Fifteen minutes after the injection, the mucosa was removed down to the masseter muscle or orbicularis oris muscle layer using a 6-mm biopsy punch. Six weeks after the operation, the buccal mucosae were harvested after euthanasia. Morphologic and histologic examinations were performed., Results: The administration of 0.5 μg/mL TGFβ3 induced rapid re-epithelialization and suppressed scar formation. In the submucosal layer, favorable restoration of hyaluronic acid and elastin was seen in the TGFβ3-treated groups compared to the saline-treated group., Conclusions: TGFβ3 is considered to be effective for better restoration of extracellular matrices of injured buccal mucosa, suggesting a preventative effect of buccal mucosa scarring., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives/hypothesis: The maturation process of scarred vocal folds has previously been investigated using animal models. However, in human models the features of scarred vocal folds have rarely been described, and the process by which the scar changes with time is not well known. The present study aimed to investigate the maturation process of human vocal folds scarred by cordectomy in terms of vibratory and aerodynamic functions., Study Design: Prospective case series., Methods: Eight patients with early glottic carcinoma and two patients with leukoplakia of the vocal fold underwent endoscopic cordectomy at Kyoto University Hospital between 2006 and 2008. The temporal changes in their vocal functions were evaluated using acoustic and aerodynamic analyses and videostroboscopic examination., Results: Normalized mucosal wave amplitude, mean flow rate, and the amplitude perturbation quotient appear to stabilize about 6 months after the procedure. Although there were individual variations in the changes in normalized glottal gap and maximum phonation time, it appears to take at least 6 months to reach plateau. The other parameters-pitch perturbation quotient and noise to harmonic ratio-varied by individual, and thus it was difficult to identify commonalities in the healing process., Conclusions: Some individual variation was observed in the temporal changes of vocal function of scarred vocal folds after cordectomy. However, in terms of vibratory and aerodynamic functions, this study suggests that it takes at least 6 months for maturation of vocal fold scarring.

Objectives/hypothesis: 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., Study Design: Animal model., Methods: Vocal folds from eight beagles were unilaterally scarred by stripping the entire layer of the lamina propria; contralateral vocal folds were kept intact as normal controls. Six months after the procedures, hydrogels (0.5 mL) containing 1 microg of HGF were injected into the scarred vocal folds of four dogs (HGF-treated group). Hydrogels containing saline solution were injected into the other four dogs (sham group). Histological and vibratory examinations on excised larynges were completed for each group 9 months after the initial surgery., Results: Experiments conducted on excised larynges demonstrated significantly better vibrations in the HGF-treated group in terms of mucosal wave amplitude. Although phonation threshold pressure was significantly lower in the HGF-treated group compared with the sham group, no significant differences were observed in the normalized glottal gap between HGF-treated and sham groups. Histological examinations of the HGF-treated vocal folds showed reduced collagen deposition and less tissue contraction with favorable restoration of hyaluronic acid., Conclusions: Results suggest that administration of HGF may have therapeutic potential in the treatment of chronic vocal fold scarring.

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.

Objective: The aim of this study was to regenerate high-quality cranial bone using tissue engineering techniques, with subsequent extension to clinical application. Our previous study with a 3-month observation period indicated that a composite scaffold composed primarily of beta-tricalcium phosphate (TCP) had the potential for cranial bone regeneration. In this study, we investigated whether bone marrow derived stromal cells (BSCs) could promote the regeneration of cranial bone as determined after 3 and 6 months., Study Design: The pilot study was conducted with 14 adult beagle dogs., Materials and Methods: Craniotomy was performed in the same manner used clinically. The bone defect (2 x 2 cm) was created at each canine temporoparietal region. The test animals were divided into three groups. In group I, the bone defect was closed by replacing the original free bone flap without filling the residual gaps. In group II, the gap was filled with a composite scaffold consisting of collagen coated beta-TCP and autologous bone fragments with fibrin glue. In group III, autologous cultured BSCs and the composite scaffold were used to fill the gap. The sites of craniotomy were analyzed with three-dimensional computed tomography and histologic examination 3 and 6 months after the operation., Results: Bone regeneration was observed in groups II and III, with more extensive formation in group III than in group II. In group I, bone regeneration was not observed., Conclusion: This study showed that BSCs have the potential to promote cranial bone regeneration and confirmed the efficacy of a composite scaffold made of beta-TCP and autologous bone fragments with fibrin glue.

Objectives/hypothesis: Our aim was to evaluate the combined effect of 5-fluorouracil (5-FU) and myringotomy for the treatment of otitis media with effusion (OME). OME is usually treated with medication, myringotomy, or insertion of a ventilation tube (VT). Except for VT insertion, however, treatment effects are short-lived. VT insertion has numerous sequelae: increased susceptibility to infection, large perforation of the tympanic membrane, cholesteatoma, and eventual hearing deterioration. We estimated the depressant action of 5-FU on normal cell proliferation in vitro. In addition, clinically, we assessed whether 5-FU has the potential to prolong the effect of myringotomy., Study Design: An in vitro study and a clinical study were conducted., Materials and Methods: In study I, fibroblasts harvested from the peritoneum of three green fluorescent protein transgenic mice were cultured with different doses of 5-FU. After 2 weeks, their proliferation rates were compared. In study II, patients (54 males, 47 females) were selected randomly from a group of patients with intractable OME. Myringotomy with or without a single dose of 5-FU ointment (approximately 0.10-0.30 mg) was performed in group I (n = 64) and group II (n = 37), respectively. The natural closure rates of the tympanic membrane were assessed in both groups., Results: In vitro, 5-FU inhibited the growth of fibroblasts in a dose-dependent manner. The average time to tympanic membrane closure was 20.5 days in group I and 8.1 days in group II. No adverse events were observed in either group., Conclusions: Topical application of 5-FU ointment is useful in prolonging the effect of myringotomy. 5-FU ointment therapy is easy, safe, and cost-effective and may be of wide application.

Objectives: To regenerate of the mastoid air cells and their functions for the treatment of incurable otitis media., Materials and Method: In situ tissue-engineered mastoid air cells using three-dimensional hydroxy apatite (3D-HA) of honeycomb-like structure were used as artificial pneumatic bones. This 3D-HA is made of calcium phosphate and has a high ratio of micropores, 90%. Its surface is coated with collagen. Ten patients (4 males, 6 females) for this clinical study were randomly selected among the patients with severe cholesteatoma, adhesive otitis media, and purulent chronic otitis media, and they were to be received a staged operation. At the first stage of tympanoplasty, collagen-coated 3D-HA was put into the newly opened mastoid cavity, and it was fixed by fibrin glue. Recovery of mastoid aeration and regeneration of the pneumatic air cells of the mastoid cavity were estimated by images of high-resolution computed tomography (HRCT) after the first operation. At the second stage of operation, histopathologic examinations of specimens of HA taken from the reopened mastoid cavity were performed., Results: Aerations in the mastoid cavity were observed in 9 of the 10 patients within 12 months after the second operation. Moreover, the pneumatic structure in the mastoid cavity was partially regenerated in five patients. In these successful cases, mucosa with newly formed capillaries was observed on the surface of the implanted HA at the second stage of operation. In the failure cases, however, connective tissues and granulations invaded the space of the 3D-HA. They were observed as soft-tissue density areas in the HRCT scan images., Conclusions: This study indicates that mucosa can grow on the surface of implanted artificial 3D-HA and can have a gas exchange function in the newly opened mastoid cavity. These tissue-engineered mastoid air cells may be a possible treatment for intractable otitis media.