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Objectives: Intraoperative cone beam computed tomography (CBCT) imaging has the potential to facilitate the surgical procedure. The current preliminary retrospective chart review investigated the benefits of intraoperative CBCT during laryngoplasty., Method: This study examined 26 cases that underwent intraoperative CBCT imaging during laryngoplasty, with one patient who counted twice due to first and revision surgery. The visual quality of structures of interest (glottal shape, thyroid cartilage, arytenoid cartilage, and implants) was determined using intraoperative CBCT during laryngoplasty. Each patient also underwent an aerodynamic assessment., Results: CBCT provided unique information, such as surgical landmarks in severe scarring, the subglottal shape, and the rotation angle of the arytenoid cartilage during arytenoid adduction. Nonetheless, 26.9% (7 of 26) of cases were affected by motion artifact, due to the long acquisition time. When motion artifact-negative cases were evaluated, 100% of glottal shape and more than 89% of thyroid cartilage were well visualized. All arytenoids were well-visualized in patients ≥ 50 years of age and without motion artifact, while CBCT failed to visualize the arytenoids in 2 of 4 patients who were < 50 years, due to the lack of calcifications. After medialization surgery, the yields of improved maximal phonation times (MPTs) in the motion artifact-negative and -positive groups were 8.7 sec and 3.4 sec, respectively (p = 0.032; Welch's t test). This comparison indicates intraoperative CBCT would contribute in MPT improvement, if CBCT is taken in measurable quality., Conclusion: The potential benefits of intraoperative CBCT during laryngoplasty were demonstrated. A corollary, prospective study is warranted to further confirmation., Competing Interests: Declaration of Competing Interest All authors participated have no financial support or relationship that causes a conflict of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions., (Copyright © 2022. Published by Elsevier B.V.)

Previous studies transplanted human-induced pluripotent stem cells (hiPSCs)-derived mesenchymal stem cells (iMSCs) into thyroid cartilage defect of X-liked severe combined immunodeficiency (X-SCID) rats and confirmed transplanted cell survival and cartilage regeneration. Thus, this study aimed to investigate the contribution of iMSC transplantation to thyroid cartilage regeneration of nude rats. iMSCs were induced from hiPSCs via a neural crest cell lineage. Then, clumps formed from an iMSC/extracellular matrix complex were transplanted into thyroid cartilage defects in nude rats. The larynx was removed and histological and immunohistochemical analyses were performed 4 or 8 weeks after the transplantation. Human nuclear antigen (HNA)-positive cells were observed in 11 of 12 (91.7%) rats, which indicated that transplanted iMSCs survived in thyroid cartilage defects in nude rats. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells in 8 of 12 rats (66.7%), which indicated cartilage-like regeneration. Cartilage-like regeneration in nude rats in this study was comparable to the previous report on X-SCID rats (HNA-positive cells were observed in all 14 rats and cartilage-like regeneration was observed in 10 of 14 rats). This result suggests that nude rats could be an alternative to X-SCID rats in thyroid cartilage regeneration experiments using iMSCs, and this nude rat cartilage transplantation model may develop cartilage regeneration research concerning fewer problems such as infection due to immunosuppression.

Introduction: Revision framework surgeries might be required for unilateral vocal fold paralyses. However, outcomes and indications of revision surgeries have not been adequately documented. For a better understanding of indications for the procedure and to help in achieving better vocal outcomes, we performed a retrospective chart review of patients who underwent revision framework surgeries for unilateral vocal fold paralysis., Objectives: This study aimed to present clinical features of patients who underwent revision framework surgeries for the treatment of unilateral vocal fold paralysis., Methods: Of the 149 framework surgeries performed between October 2004 and October 2019, 21 revision framework surgeries were performed in 19 patients. Self-assessments by patients using the voice handicap index-10 questionnaire, and objective aerodynamic and acoustic assessments performed pre- and post-operatively were analyzed using the Wilcoxon's signed-rank test for paired comparisons., Results: Undercorrection was indicated as reasons for revision surgeries in all cases. The revision techniques included type I thyroplasty, type IV thyroplasty, and arytenoid adduction, and revision surgeries were completed without any severe complication in all cases. Pre- and post-operative voice handicap index-10 scores were obtained in 12 cases, and other parameters were evaluated in 18 cases. Significant improvements were observed in voice handicap index-10 scores, maximum phonation time, mean flow rate, Current/Direct Current ratio, and pitch perturbation quotient., Conclusion: Undercorrection was observed in all patients who underwent revision framework surgeries for unilateral vocal fold paralysis, and the initial assessment and planning are thought to be important in order to avoid revision surgeries. Revision surgeries were performed safely in all cases, and significantly improved vocal outcomes were observed, even after multiple procedures. Revision surgery should be considered for patients with unsatisfactory vocal functions after primary framework surgeries for unilateral vocal fold paralysis., (Copyright © 2020 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.)

Prophylactic elective neck dissection (ND) with navigation surgery using radioisotope-based sentinel lymph node biopsy (SLNB) is non-inferior to elective ND in terms of survival but has an advantage in postoperative functional disability. We conducted a subgroup analysis to identify predictive factors for false-negative (FN)-SLNB in patients with early oral cavity cancer. This study is a supplementary analysis using the dataset of a previously reported randomized clinical trial on SLN navigation surgery for oral cancers. This study investigated the association of clinical and SLN-related factors with false-negative cases in the SLNB group. From 2011 to 2016, 275 patients were enrolled and randomly assigned to the ND and SLNB study groups, with 134 patients assigned to the SLNB group. In the SLNB group, seven cases with negative SLNs and neck recurrences were judged as FN-SLNBs according to the general definition. The number of detected SLNs with and without adjusting for the propensity score was significantly associated with FNs in the logistic analysis. FN-SLNB was associated with the number of identified SLNs, suggesting the need for careful postoperative monitoring for neck recurrence in patients with one or two identified SLNs after acquiring sufficient experience in the identification technique., (© 2022. The Author(s).)

Vocal fold (VF) fibrosis is a major cause of intractable voice-related disability and reduced quality of life. Excision of fibrotic regions is suboptimal and associated with scar recurrence and/or further iatrogenic damage. Non-surgical interventions are limited, putatively related to limited insight regarding biochemical events underlying fibrosis, and downstream, the lack of therapeutic targets. YAP/TAZ integrates diverse cell signaling events and interacts with signaling pathways related to fibrosis, including the TGF-β/SMAD pathway. We investigated the expression of YAP/TAZ following vocal fold injury in vivo as well as the effects of TGF-β1 on YAP/TAZ activity in human vocal fold fibroblasts, fibroblast-myofibroblast transition, and TGF-β/SMAD signaling. Iatrogenic injury increased nuclear localization of YAP and TAZ in fibrotic rat vocal folds. In vitro, TGF-β1 activated YAP and TAZ in human VF fibroblasts, and inhibition of YAP/TAZ reversed TGF-β1-stimulated fibroplastic gene upregulation. Additionally, TGF-β1 induced localization of YAP and TAZ in close proximity to SMAD2/3, and nuclear accumulation of SMAD2/3 was inhibited by a YAP/TAZ inhibitor. Collectively, YAP and TAZ were synergistically activated with the TGF-β/SMAD pathway, and likely essential for the fibroplastic phenotypic shift in VF fibroblasts. Based on these data, YAP/TAZ may evolve as an attractive therapeutic target for VF fibrosis.

The laryngotracheal cartilage is a cardinal framework for the maintenance of the airway for breathing, which occasionally requires reconstruction. Because hyaline cartilage has a poor intrinsic regenerative ability, various regenerative approaches have been attempted to regenerate laryngotracheal cartilage. The use of autologous mesenchymal stem cells (MSCs) for cartilage regeneration has been widely investigated. However, long-term culture may limit proliferative capacity. Human-induced pluripotent stem cell-derived MSCs (iMSCs) can circumvent this problem due to their unlimited proliferative capacity. This study aimed to investigate the efficacy of iMSCs in the regeneration of thyroid cartilage in immunodeficient rats. Herein, we induced iMSCs through neural crest cell intermediates. For the relevance to prospective future clinical application, induction was conducted under xeno-free/serum-free conditions. Then, clumps fabricated from an iMSC/extracellular matrix complex (C-iMSC) were transplanted into thyroid cartilage defects in immunodeficient rats. Histological examinations revealed cartilage-like regenerated tissue and human nuclear antigen (HNA)-positive surviving transplanted cells in the regenerated lesion. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells. These results indicated that the transplanted C-iMSCs promoted thyroid cartilage regeneration and some of the iMSCs differentiated into chondrogenic lineage cells. Induced MSCs may be a promising candidate cell therapy for human laryngotracheal reconstruction., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Multiciliated epithelial cells in the airway are essential for mucociliary clearance. Their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. Epithelial surfaces were observed using an epi-fluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.

Objective: We hypothesize that Smad3 is a master regulator of fibrosis in the vocal folds (VFs) and RNA-based therapeutics targeting Smad3 hold therapeutic promise. Delivery remains challenging. We previously described a novel synthetic peptoid oligomer, lipitoid L0 , complexed with siRNA to improve stability and cellular uptake. An advantage of these peptoids, however, is tremendous structural and chemical malleability to optimize transfection efficiency. Modifications of L0 were assayed to optimize siRNA-mediated alteration of gene expression., Methods: In vitro, Smad3 knockdown by various lipitoid variants was evaluated via quantitative real-time polymerase chain reaction in human VF fibroblasts. Cytotoxicity was quantified via colorimetric assays. In vivo, a rabbit model of VF injury was employed to evaluate the temporal dynamics of Smad3 knockdown following injection of the L0 -siRNA complex., Results: In vitro, similar reductions in Smad3 expression were established by all lipitoid variants, with one exception. Sequence variants also exhibited similar nontoxic characteristics; no statistically significant differences in cell proliferation were observed. In vivo, Smad3 expression was significantly reduced in injured VFs following injection of L0 -complexed Smad3 siRNA at 1 day postinjection. Qualitative suppression of Smad3 expression persisted to 3 days following injury, but did not achieve statistical significance., Conclusions: In spite of the chemical diversity of these peptoid transfection reagents, the sequence variants generally provided consistently efficient reductions in Smad3 expression. L0 yielded effective, yet temporally limited knockdown of Smad3 in vivo. Peptoids may provide a versatile platform for the discovery of siRNA delivery vehicles optimized for clinical application., Level of Evidence: NA., (© 2019 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals, Inc. on behalf of The Triological Society.)

Objectives/hypothesis: Direct glucocorticoid (GC) injection for vocal fold (VF) scarring has evolved as a therapeutic strategy, but the mechanisms underlying the antifibrotic effects remain unclear. GCs act via the glucocorticoid receptor (GR), which is phosphorylated at multiple serine residues in a hormone-dependent manner to affect bioactivity. We hypothesize that GCs regulate SMAD signaling via GR phosphorylation in vocal fold fibroblasts (VFFs)., Study Design: In vitro., Methods: Human VFFs were treated with dexamethasone (DM; 10 -5 -10 -7 M) ± transforming growth factor (TGF)-β1 (10 ng/mL). RU486 (10 -6 M) was employed to isolate the regulatory effects of GR. Total GR, Ser 211 , and Ser 203 phosphorylation was examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunocytochemistry. Quantitative polymerase chain reaction was employed to determine GR-mediated effects of DM on genes related to fibrosis., Results: Total GR and Ser 211 phosphorylation was observed predominantly in the nucleus 1 hour after DM administration. DM decreased total GR expression, but Ser 203 and Ser 211 phosphorylation increased. RU486 limited the effects of DM. SMAD3 and SMAD7 mRNA expression significantly decreased 4 hours after DM administration (P < 0.05); this response was negated by RU486. COL1A1 remained unchanged, and ACTA2 significantly increased following 24 hours of DM treatment (P < 0.05)., Conclusion: DM regulated TGF-β1 signaling via altered SMAD3 and SMAD7 expression. This response was associated with altered GR phosphorylation. These findings provide insight into the mechanisms of steroidal effects on vocal fold repair; ultimately, we seek to enhance therapeutic strategies for these challenging patients., Level of Evidence: NA Laryngoscope, 129:E187-E193, 2019., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: To elucidate the aging physiology of the vocal folds, we examined the characters of aged vocal fold fibroblasts (VFFs) in various conditions., Study Design: In vitro study., Methods: VFFs from young (12-week-old) and aged (19-month-old) Sprague-Dawley rats were compared. Proliferative capacity, ratio of myofibroblast to fibroblast, myofibroblast function, and extracellular matrix production were examined in the following conditions: naïve, basic fibroblast growth factor (bFGF) supplemented, and hepatocyte growth factor (HGF) supplemented., Results: Aged VFFs demonstrated reduced proliferation by cell counting, though the ratio of Ki-67-positive cells showed no difference. Aged VFFs exhibited an increased expression of α-smooth muscle actin (α-SMA); however, they demonstrated no enhanced contractile ability in a gel contraction assay. Type I collagen protein was increased age dependently, accompanied with decreased Mmp1 and unchanged Col1a1 transcription. Type I collagen protein and α-SMA represented quite similar reduction patterns to bFGF or HGF administration., Conclusions: The following possible characteristics of aged VFFs were implied: long duration of mitosis, increased myofibroblast population size with certain dysfunctions, reduced type I collagen turnover, and correlation between α-SMA expression and type I collagen metabolism. Further investigations of these features will help to clarify presbyphonia's pathology and establish treatment strategies., Level of Evidence: NA Laryngoscope, 129:E94-E101, 2019., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Our laboratory recently described NR4A1 as an endogenous inhibitor of TGF-β-induced vocal fold (VF) fibrosis. Our prior report described the temporal expression of NR4A1 during VF healing in vivo and the effects of NR4A1 knockdown on fibroplastic cell activities in vitro. Based on these findings, we hypothesized that cytosporone-B (Csn-B), an NR4A1 agonist, may hold significant therapeutic potential., Study Design: In vitro., Methods: Human VF fibroblasts were exposed to TGF-β1+/-Csn-B. Expression of genes related to fibrosis were quantified. In addition, contraction was assayed as a surrogate for the fibrotic phenotype in our cell line., Results: TGF-B1 stimulated COL1A1 and ACTA2, as expected. Csn-B significantly downregulated TGF-β1-mediated upregulation of these genes (P = .009, P = .03, respectively). Csn-B had no effect on genes related to TGF-β/Smad signaling. Csn-B also decreased the TGF-β1-mediated contractile phenotype in our cells (P = .004)., Conclusions: NR4A1 is an endogenous inhibitor of fibrosis in the vocal folds and Csn-B, as an NR4A1 agonist, may evolve as an ideal, therapeutic candidate for this challenging condition., Level of Evidence: NA Laryngoscope, 128:E425-E428, 2018., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: To develop a clinically relevant model of oropharyngeal concurrent chemoradiation therapy (CCRT) in order to quantify the effects of CCRT on tongue function and structure. CCRT for advanced oropharyngeal cancer commonly leads to tongue base dysfunction and dysphagia. However, no preclinical models currently exist to study the pathophysiology of CCRT-related morbidity, thereby inhibiting the development of targeted therapeutics., Study Design: Animal model., Methods: Twenty-one male Sprague-Dawley rats were randomized into three groups: 2 week (2W), 5 month (5M), and control (C). The 2W and 5M animals received cisplatin, 5-fluorouracil, and five fractions of 7 Gy to the tongue base; the C animals received no intervention. In vivo tongue strength and displacement, as well as hyoglossus muscle collagen content, were assessed. Analyses were conducted 2 weeks or 5 months following completion of CCRT in the 2W and 5M groups, respectively., Results: Peak tetanic and twitch tongue forces were significantly reduced in both 2W and 5M animals compared to controls (tetanic: P = .0041, P = .0089, respectively; twitch: P = .0201, P = .0020, respectively). Twitch half-decay time was prolonged in 2W animals compared to controls (P = .0247). Tongue displacement was significantly reduced across all testing parameters in 5M animals compared to both the C and 2W groups. No differences in collagen content were observed between experimental groups., Conclusions: The current study is the first to describe a preclinical model of CCRT to the head and neck with an emphasis on clinical relevance. Tongue strength decreased at 2 weeks and 5 months post-CCRT. Tongue displacement increased only at 5 months post-CCRT. Fibrosis was not detected, implicating alternative causative factors for these findings., Level of Evidence: NA Laryngoscope, 1783-1790, 2018., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Our laboratory and others hypothesized that Smad3 is a principle mediator of the fibrotic phenotype in the vocal folds (VFs), and we further posited that alteration of Smad3 expression through short interfering (si)RNA holds therapeutic promise, yet delivery remains challenging. To address this issue, we employed a novel synthetic oligomer, lipitoid, complexed with siRNA to improve stability and cellular uptake with the goal of increased efficiency of RNA-based therapeutics., Study Design: In vitro study and in vivo animal model., Methods: In vitro, lipitoid cytotoxicity was quantified via colorimetric and LIVE/DEAD assays in immortalized human VF fibroblasts and primary rabbit VF fibroblasts. In addition, optimal incubation interval and solution for binding siRNA to lipitoid for intracellular delivery were determined. In vivo, a rabbit model of VF injury was employed to evaluate Smad3 knockdown following locally injected lipitoid-complexed siRNA., Results: In vitro, lipitoid did not confer additional toxicity compared to commercially available reagents. In addition, 20-minute incubation in 1× phosphate-buffered saline resulted in maximal Smad3 knockdown. In vivo, Smad3 expression increased following VF injury. This response was significantly reduced in injured VFs at 4 and 24 hours following injection (P = .035 and .034, respectively)., Conclusions: The current study is the first to demonstrate targeted gene manipulation in the VFs as well as the potential utility of lipitoid for localized delivery of genetic material in vivo. Ideally, these data will serve as a platform for future investigation regarding the functional implications of therapeutic gene manipulation in the VFs., Level of Evidence: NA. Laryngoscope, 128:E178-E183, 2018., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

Vocal fold scar and sulcus are intractable diseases with no effective established treatments. Hepatocyte growth factor (HGF) has preclinically proven to have potent antifibrotic and regenerative effects on vocal fold scar. The current Phase I/II clinical trial aims to examine the safety and effectiveness of intracordal injection of a recombinant human HGF drug for patients with vocal fold scar or sulcus. This is an open-label, dose-escalating, first-in-human clinical trial. Eighteen patients with bilateral vocal fold scar or sulcus were enrolled and divided into three groups: Step I received 1 μg of HGF per vocal fold; Step II received 3 μg of HGF; and Step III received 10 μg of HGF. Injections were administered once weekly for 4 weeks. The protocol treatment was performed starting with Step I and escalating to Step III. Patients were followed for 6 months post-treatment. Local and systemic safety aspects were examined as primary endpoints, and therapeutic effects were assessed as secondary endpoints using voice handicap index-10; maximum phonation time; vocal fold vibratory amplitude; grade, rough, breathy, asthenic, strained scale; and jitter. The results indicated no serious drug-related adverse events in either the systemic or local examinations. In whole-subject analysis, voice handicap index-10, vocal fold vibratory amplitude, and grade, rough, breathy, asthenic, strained scale were significantly improved at 6 months, whereas maximum phonation time and jitter varied. There were no significant differences in phonatory data between the step groups. In conclusion, intracordal injection of a recombinant human HGF drug was safe, feasible, and potentially effective for human patients with vocal fold scar or sulcus., (Copyright © 2017 John Wiley & Sons, Ltd.)

Objectives/hypothesis: This study aimed to reveal the effects of adipose-derived mesenchymal stromal cells (ASCs) on prevention of vocal fold scarring by investigating how the immediate ASCs transplantation into the injured rat vocal fold affect the levels of gene transcription and translation., Study Design: Prospective animal experiments with controls., Methods: ASCs harvested from green fluorescent protein transgenic rat (ASCs group) or saline (sham group) were injected into the thyroarytenoid muscle of Sprague-Dawley rats immediately after stripping the vocal fold. For histological examinations, larynges were extirpated at 3, 14, and 56 days after the injection. Quantitative real-time polymerase chain reaction (PCR) analyses were performed at 3 and 14 days after the injection., Results: Transplanted ASCs were detected only in larynges at day 3. At days 14 and 56, histological examination showed significantly higher amounts of hyaluronic acid and lower deposition of collagen in the ASCs group compared to the sham group. Real-time PCR revealed that the ASCs group showed low expression of procollagen (Col)1a1, Col1a3, matrix metalloproteinase (Mmp)1 and Mmp8 in each time points. The ASCs group showed high expression of fibroblast growth factor (Fgf)2 and Hepatocyte growth factor (Hgf) compared to the sham group at day 14., Conclusions: ASCs increased expressions of Fgf2 and Hgf, and suppressed excessive collagen deposition during vocal fold wound healing. Given the fact that ASCs survived no more than 14 days, ASCs were thought to induce upregulations of growth factors' genes in surrounding cells. These results suggested that ASCs have potential to prevent vocal fold scarring., Level of Evidence: NA. Laryngoscope, 128:E33-E40, 2018., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

Objective: Recent reports highlight the efficacy of small interfering RNA (siRNA) targeting SMAD3 to regulate transforming growth factor β (TGF-β)-mediated fibroplasia in vocal fold fibroblasts. The current study sought to investigate SMAD3 expression during wound healing in vivo and quantify the downstream transcriptional events associated with SMAD3 knockdown in vitro., Study Design: In vivo and in vitro., Methods: Unilateral vocal fold injury was created in a rabbit model. SMAD3 and SMAD7 mRNA expression was quantified at 1 hour and 1, 3, 7, 14, 30, 60, and 90 days following injury. In vitro, multi-gene analysis technology was employed in our immortalized human vocal-fold fibroblast cell line following TGF-β1 stimulation ± SMAD3 knockdown across time points., Results: SMAD3 mRNA expression increased following injury; upregulation was significant at 3 and 7 days compared to control (both P < 0.001). SMAD7 mRNA was also upregulated at 3, 7, and 14 days (P = 0.02, P < 0.001, and P < 0.001, respectively). In vitro, SMAD3 knockdown reduced the expression of multiple profibrotic, TGF-β signaling, and extracellular matrix metabolism genes at 6 and 24 hours following TGF-β1 stimulation., Conclusion: Cumulatively, these data support SMAD3 as a potential master regulator of TGF-β-mediated fibrosis. SMAD3 transcription peaked 7 days following injury. Multi-gene analysis indicated that the therapeutic effectiveness of SMAD3 knockdown may be related to regulation of downstream mediators of fibroplasia and altered TGF-β signaling., Level of Evidence: NA. Laryngoscope, 127:E308-E316, 2017., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: NR4A1 was recently identified as an endogenous inhibitor of transforming growth factor (TGF)-β-induced fibrosis, and the role of this nuclear receptor has not been elucidated in tissue health or the response to injury in the vocal folds. Given the clinical implications of vocal fold fibrosis, we investigated NR4A1 expression during vocal fold wound healing in vivo and the regulatory roles of NR4A1 on vocal fold fibroblasts (VFFs) in vitro with the ultimate goal of developing targeted therapies for this challenging patient population., Study Design: In vivo and in vitro., Methods: In vivo, the temporal pattern of NR4A1 mRNA expression was quantified following rat vocal fold injury. In vitro, the role of NR4A1 on TGF-β1-mediated transcription of genes underlying fibrosis as well as myofibroblast differentiation and collagen gel contraction was quantified in our human VFF line. Small interfering RNA was employed to alter NR4A1 expression to further elucidate this complex system., Results: Nr4a1 mRNA increased 1 day after injury and peaked at 7 days. Knockdown of NR4A1 resulted in upregulation of COL1A1 and TGF-β1, with TGF-β1 stimulation (both P < .001) in VFFs. NR4A1 knockdown also resulted in increased α-smooth muscle actin-positive cells (P = .013) and contraction (P = .002) in response to TGF-β1., Conclusions: NR4A1 has not been described in vocal fold health or disease. Upregulation of TGF-β following vocal fold injury was concurrent with increased NR4A1 expression. These data provide a foundation for the development of therapeutic strategies given persistent TGF-β signaling in vocal fold fibrosis., Level of Evidence: N/A Laryngoscope, 127:E317-E323, 2017., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: Interactions between mesenchymal stem cells (MSCs) and native vocal fold fibroblasts (VFFs) have not been described in spite of promising preliminary data regarding the effects of MSCs on vocal fold repair in vivo. The current study employed a conditioned media (CM) model to investigate the paracrine effects of bone marrow-derived mesenchymal stem cells (BMSCs) on VFFs., Methods: Human VFFs were treated with transforming growth factor-β1 (TGF-β1; 10 ng/mL), CM from human BMSCs following 48 hours of TGF-β1 stimulation, or CM+TGF-β1. Proliferation, immunocytochemistry for alpha smooth muscle actin (αSMA), migration, and collagen gel contraction were quantified as well as transcription of components of the TGF-β signaling pathway., Results: Transforming growth factor-β1 accelerated proliferation and induced αSMA in VFFs; these effects were suppressed with CM ( P = .009, P < .001, respectively). The CM+TGF-β1 condition increased cell migration ( P = .02) and decreased gel contraction; CM+TGF-β1 also inhibited TGF-β signaling via significant upregulation of NR4A1 as well as downregulation of S MAD3 and TGF-β1 relative to TGF-β1 stimulation in the absence of CM ( P = .002, P < .001, and P = .005, respectively)., Conclusions: Conditioned media affected many profibrotic cell activities in TGF-β1-stimulated VFFs, likely related to altered TGF-β signaling. These data provide preliminary insight regarding the antifibrotic effects of MSCs and further support their progression to clinical utility.

Objective: An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a lipitoid transfection reagent in comparison to a commercially available reagent., Study Design: In vitro., Methods: We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the transforming growth factor-β cascade implicated in fibroplasia in the vocal folds and other tissues., Results: The two reagents suppressed Smad3 mRNA for up to 96 hours; lipitoid performed favorably and comparably. Both compounds yielded 60% to 80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (P < 0.05 relative to control). Dose and number of administrations played a significant role in gene suppression (P < 0.05). Suppression was more dose-sensitive with lipitoid. At a constant siRNA concentration, a 50% decrease in gene expression was observed in response to a five-fold increase in lipitoid concentration. Increased number of administrations enhanced gene suppression, ∼45% decrease between one and four administrations. Neither serum nor media type altered efficiency., Conclusion: Lipitoid effectively knocked down Smad3 expression across multiple transfection conditions. These preliminary data are encouraging, and lipitoid warrants further investigation with the goal of clinical utility., Level of Evidence: NA. Laryngoscope, 127:E231-E237, 2017., (© 2016 The American Laryngological, Rhinological and Otological Society, Inc.)

Vocal fold scar remains a therapeutic challenge. Basic fibroblast growth factor (bFGF) was reported to have regenerative effects for vocal fold scar, although it has the disadvantage of rapid absorption in vivo. A collagen-gelatin sponge (CGS) can compensate for the disadvantage by providing a sustained release system. The current study evaluated the efficacy of CGS combined with bFGF on vocal fold scar, using rat fibroblasts for an in vitro model and a canine in vivo model. We prepared fibroblasts from scarred vocal folds (sVFs) in rats and showed that bFGF accelerated cell proliferation and suppressed expression levels of cleaved caspase 3 and α-smooth muscle actin. Has 1, Has 3, Fgf2, Hgf and Vegfa mRNA levels were significantly upregulated, while Col1a1 and Col3a1 were dose-dependently downregulated, with a maximum effect at 100 ng/ml bFGF. In an in vivo assay, 6 weeks after lamina propria stripping, beagles were divided into three groups: CGS alone (CGS group); CGS with bFGF (7 µg/cm 2 ; CGS + bFGF group); or a sham-treated group. Vibratory examination revealed that the glottal gap was significantly reduced in the bFGF group and the two implanted groups, whereas the CGS + bFGF group showed higher mucosal wave amplitude. Histological examination revealed significantly restored hyaluronic acid and elastin redistribution in the CGS + bFGF group and reductions in dense collagen deposition. These results provide evidence that CGS and bFGF combination therapy may have therapeutic potential and could be a promising tool for treating vocal fold scar. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Objective Given the recalcitrant nature of recurrent respiratory papillomatosis, targeted therapies to reduce disease burden are fundamental to improved patient care paradigms. We seek to demonstrate the safety of imiquimod injection into vocal fold mucosa by evaluating the degree of laryngeal edema, histopathologic changes to vocal fold structure, and serologic interferon α (IFNα) levels following injection. Study Design Preclinical. Setting Academic institution. Subjects and Methods Six New Zealand White rabbits underwent unilateral injection of 100 µg of sterile imiquimod (1 µg/µL), with 100 µL of normal saline injected into the contralateral vocal fold. Direct laryngoscopy was performed on days 3, 7, and 30 following injection. Larynges from 3 rabbits were harvested on postinjection day 7 for histologic analysis. The remaining 3 rabbit larynges were harvested on day 30. Serial serum samples were drawn for IFNα quantification via immunoassay. Results No signs of respiratory distress were observed at any point. Vocal fold appearance was not clinically divergent between imiquimod and control conditions via serial direct laryngoscopic evaluation. No inflammatory lesions or scarring were identified following injection. Histology showed no signs of acute inflammatory processes or changes in the control or imiquimod injection groups. Serum IFNα increased at days 3 and 7 following imiquimod injection ( P < .0001 and P = .0368, respectively), before returning to baseline by day 14. Conclusions Vocal fold imiquimod injection did not result in notable morbidity in this preclinical model. However, serum IFNα concentrations increased transiently. These data are critical to advance the therapeutic utility of this compound, particularly in the setting of recurrent respiratory papillomatosis.

Objective: There continue to be therapeutic challenges in the management of vocal fold scarring. We previously showed that basic fibroblast growth factor (bFGF) injection has therapeutic potential for vocal fold scarring. However, the working time of bFGF is relatively short, and multiple injections were required in many cases to obtain the regenerative effect. An efficacious delivery system for bFGF has yet to be established. We designed a method of sustained drug delivery system (DDS) of bFGF by using a gelatin hydrogel. Hydrogel has been developed for targeted delivery and controlled release of bFGF. Hydrogel of the particle type is also injectable and commercially available. The current study aims to investigate the effects of a single injection of bFGF-DDS on acute vocal fold scarring using a canine model., Methods: Vocal folds from eight beagles were unilaterally scarred by stripping the lamina propria. One month later, hydrogels (0.5ml) containing 10μg of bFGF were injected into the scarred vocal folds of four beagles (FGF-hydrogel group). Saline (0.5ml) was injected into the other four beagles (sham group). Vibratory and histological examination of excised larynges was performed 5 months after treatment. Comparative analysis between the current data and our previous data with repeated injection of bFGF solution was also completed., Results: Vibratory examination demonstrated significantly improved vibration in the bFGF hydrogel-treated group. Histological examination of the bFGF hydrogel group showed restoration of hyaluronic acid in the lamina propria as compared to sham. Comparison between the DDS system and our previous bFGF solution injection indicated better effects of the DDS system on vibratory amplitude., Conclusion: A single injection of bFGF hydrogel has regenerative effects on acute vocal fold scarring, which is at least similar to repeated injection of bFGF solution., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Objectives/hypothesis: Vocal fold scarring, which causes severe hoarseness, is intractable. The optimal treatment for vocal fold scarring has not been established; therefore, prevention of scarring is important. The aim of this study was to clarify the effectiveness of basic fibroblast growth factor (bFGF) for prevention of postsurgical vocal fold scarring., Study Design: Prospective animal experiments with controls., Method: The vocal folds of Sprague-Dawley rats were injured unilaterally or bilaterally after local application of a 10 μL solution of bFGF. Larynges ware harvested for histological and immunohistochemical examination 2 months postoperation and for quantitative real-time polymerase chain reaction (qRT-PCR) analysis 1 week postoperation., Results: Histological examination showed significantly increased hyaluronic acid and decreased deposition of dense collagen in the bFGF-treated group at 100 ng/10 μL compared with the sham-treated group. Immunohistochemical examination showed significantly decreased collagen type III deposition in the bFGF-treated group at 100 ng/10 μL compared with the sham-treated group. qRT-PCR revealed that hyaluronan synthase 2 (Has2), Has3, and hepatocyte growth factor were upregulated in bFGF-treated groups compared with sham-treated group., Conclusion: The current results suggest that local application of bFGF at the time of injury has the potential to prevent vocal fold scarring. Preventive injection of bFGF could be applied at the time of phonomicrosurgery to avoid postoperative scar formation., Level of Evidence: N/A. Laryngoscope, 2016 127:E67-E74, 2017., (© 2016 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Mesenchymal stem cells (MSCs) hold therapeutic promise for vocal fold scar, yet the precise mechanism(s) underlying tissue level changes remain unclear. We hypothesize that MSCs interact with native fibroblasts to favorably affect healing. Furthermore, we hypothesize that these interactions vary based on MSC source., Methods: Vocal fold fibroblasts (VFFs), adipose-derived stem cells, and bone marrow-derived stem cells (BMSCs) were extracted from Sprague-Dawley rats; and a coculture model was employed culturing VFFs ± transforming growth factor (TGF-β1) (10 ng/mL) ± MSCs. Monoculture MSCs were also prepared as a control. Both extracellular matrix (ECM) and components of the TGF-β signaling pathway were analyzed via polymerase chain reaction and western blotting., Results: Significantly decreased TGF-β1 mRNA and α-smooth muscle actin protein was observed in VFFs in response to TGF-β1 in the coculture with both MSCs (P < 0.05, P < 0.01). BMSCs significantly downregulated collagen I (P < 0.05), collagen III (P < 0.05), Smad3 (P < 0.01), and TGF-β1 receptor I (P < 0.01) mRNA in VFFs. Hyaluronic synthase-1 and 2 increased in cocultured BMSCs when compared with monocultured BMSCs at baseline and in response to TGF-β1 (P < 0.01)., Conclusion: MSCs had a favorable effect on ECM regulation as well as suppression of TGF-β1 signaling in VFF. Bidirectional paracrine signaling was also observed as VFFs altered ECM regulation in MSCs. These data provide insight into the regenerative effects of MSCs and provide a foundation for clinical application., Level of Evidence: NA Laryngoscope, 127:E35-E41, 2017., (© 2016 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Inhalation injury significantly increases morbidity and mortality in burn patients. Approximately one in five burn patients have acute injury to the larynx, trachea, and/or lungs-and as many as 70% have long-term laryngeal abnormalities. Although inhalation injury to the lung has been studied extensively, no models exist to study these insults to the larynx. As such, we developed an in vivo rabbit model to create precise and reproducible laryngeal burn with resultant tissue damage as a foundation for interventional studies., Methods: Following tubeless tracheotomy, a custom temperature-control device was employed to apply heated air (70°C-80°C, 150°C-160°C, or 310°C-320°C) ± smoke derived from unbleached cotton to the larynx, endoscopically, minimizing adjacent tissue damage in six rabbits. Pain, nutrition, and level of activity were monitored. Direct laryngoscopy and histological examination were performed 24 hours following insult., Results: All animals survived injury with appropriate pain control; oral intake was initiated and all were adequately ventilating via tracheostomy. Burn sequelae were noted under direct visualization 24 hours after injury, and graded levels of edema and tissue damage were observed as a function of temperature. Edema obstructed true vocal fold visualization at increased temperatures. These injury patterns correlated with graded tissue damage on histology., Conclusion: We created an in vivo model of laryngeal burn injury employing a custom burn device resulting in graded tissue injury. This model is critical for investigation of the mechanisms underlying burn injury, and ultimately, the development and evaluation of therapies for this challenging population., Level of Evidence: NA Laryngoscope, 127:186-190, 2017., (© 2016 The American Laryngological, Rhinological and Otological Society, Inc.)

In the management of laryngeal pre-cancerous lesions such as dysplasia or carcinoma in situ (CIS), it is important that lesion regression occur without any complications. As a minimally invasive treatment option, photocoagulation therapy using angiolytic lasers has been attracting attention. Therapeutic effects have been reported for this type of treatment, however, vocal function after treatment has not been well discussed. In this retrospective case series, we examined the therapeutic effects of photocoagulation therapy on laryngeal dysplasia and the impact on vocal function. Twenty-four patients with laryngeal dysplasia or CIS were treated with photocoagulation therapy using angiolytic lasers. Two patients were treated under general anesthesia, the remaining 22 patients were treated with topical anesthesia. Before and after treatment the extent of the lesion and vocal function was evaluated by endoscopic examination and acoustic and aerodynamic analyses, respectively. More than 50 % disease regression was observed in 20 of 24 patients. Acoustic and aerodynamic analyses revealed improvement in pitch perturbation quotient with no impairment in other parameters. Photocoagulation therapy using angiolytic lasers has proven to be feasible and safe for the treatment of laryngeal dysplasia.

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: Endoscopic laryngopharyngeal surgery (ELPS) is a minimally invasive transoral surgery that was developed to treat superficial larygo-pharyngeal cancer, in which a mucosal lesion is resected transorally while preserving deeper structures by subepithelial injection. The purpose of this retrospective study is to evaluate voice outcome in patients who underwent ELPS for superficial hypopharyngeal cancer. As important structures in producing voice, such as intrinsic laryngeal muscles, their fascia, and recurrent laryngeal nerve, are located in the medial side of the piriform sinus and the postcricoid region of the hypopharynx, we focused on patients with cancer lesions involving these regions., Methods: From April 2010 to March 2011, 25 consecutive patients with superficial laryngopharyngeal cancer were treated with ELPS at Kyoto University Hospital. Among the 25 patients, 11 patients with cancer lesions on the medial side of the piriform sinus or the postcricoid area were studied. Preoperative and postoperative voice functions including maximum phonation time (MPT), mean flow rate (MFR), jitter, shimmer, soft phonation index (SPI), and noise-to-harmonic ratio (NHR), were compared retrospectively., Results: Five of 11 cancer lesions had submucosal invasion and no lesion had invaded the muscular layer pathologically. T stage was classified as Tis in 5 cases, T1 in 4 cases, and T2 in 2 cases. All lesions involved the medial side of the piriform sinus and 2 also involved the postcricoid area. Vocal fold movement was normal in all cases after the surgery. Average preoperative and postoperative values for MPT, MFR, jitter, shimmer, SPI, and NHR, were 22.7 seconds and 23.4 seconds, 165 mL/sec and 150 mL/sec, 1.53% and 1.77%, 3.82% and 5.17%, 35.5 and 36.6, and 0.13% and 0.14%, respectively. There was no statistical difference between preoperative and postoperative data for all values examined., Conclusion: ELPS is useful in preserving voice function in the treatment of superficial hypopharyngeal cancer. Preserving the deeper structures including intrinsic muscles and their fascia may be important for preserving voice function as long as the lesions are superficial.

Objective: The microflap technique is a standard procedure for the treatment of vocal fold polyps. Angiolytic laser surgery carried out under topical anesthesia is an alternative method for vocal polyp removal. However, it is not clear whether angiolytic laser surgery has the same effects on vocal outcomes as the microflap technique because of a lack of studies comparing both procedures. In the current study, vocal outcomes after both procedures were compared to clarify the effects of angiolytic laser surgery for vocal polyp removal., Methods: Vocal outcomes were reviewed for patients who underwent angiolytic laser surgery (n=20, laser group) or microflap surgery (n=34, microflap group) for vocal polyp removal. The data analyzed included patient and lesion characteristics, number of surgeries required for complete resolution, and aerodynamic and acoustic examinations before and after surgery., Results: In the laser surgery group, complete resolution of the lesion was achieved with a single procedure in 17 cases (85%) and with two procedures in 3 cases (15%). Postoperative aerodynamic and acoustic parameters demonstrated significant improvement compared to preoperative parameters in both the laser surgery group and the microflap surgery group. There were no significant differences in any postoperative aerodynamic and acoustic parameters between the two groups., Conclusion: The current retrospective study demonstrated that angiolytic laser surgery achieved complete resolution of vocal polyps within two procedures. Postoperative effects on aerodynamic and acoustic functions were similar to those after microflap surgery., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objectives: Age-related voice change is characterized as weak, harsh, and breathy. These changes are caused by histologic alteration of the lamina propria of the vocal fold mucosa as well as atrophy of the thyroarytenoid muscle. Several therapeutic strategies involving laryngeal framework surgery and injection laryngoplasty have been tried, but effects have been limited. Vocal function exercises (VFE) have been used to treat age-related vocal fold atrophy although the effectiveness has been shown with limited analysis. The present study aims to determine the effectiveness of VFE for the treatment of aged atrophy using multidimensional analysis., Study Design: This is a retrospective study., Methods: Sixteen patients with vocal fold atrophy aged 65-81 years underwent voice therapy using VFE. Six patients with vocal fold atrophy aged 65-85 years were involved as a historical control group. The grade, roughness, breathiness, asthenia, strain (GRBAS) scale, stroboscopic examinations, aerodynamic assessment, acoustic analysis, and Voice Handicap Index-10 (VHI-10) were performed before and after VFE. Normalized mucosal wave amplitude (NMWA), normalized glottal gap (NGG), and bowing index (BI) were measured by image analysis during stroboscopic examinations., Results: After VFE, significant improvements were shown in GRBAS, maximum phonation time, jitter, NMWA, NGG, and VHI-10 although BI has not changed significantly. There were no significant improvements in the historical control., Conclusions: The data suggest that VFE produces significant improvement in subjective, objective, and patient self-evaluation and deserves further attention as a treatment for aged atrophy of the vocal fold. It was also suggested that VFE does not improve the vocal fold bowing but may improve muscular function during voicing., (Copyright © 2015 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Recently in Japan, the morbidity of colorectal polyp has been increasing. As a result, a large number of cases of colorectal polyps that are diagnosed and treated using colonoscopy has now increased, and clinical guidelines are needed for endoscopic management and surveillance after treatment., Methods: Three committees [the professional committee for making clinical questions (CQs) and statements by Japanese specialists, the expert panelist committee for rating statements by the modified Delphi method, and the evaluating committee by moderators] were organized. Ten specialists for colorectal polyp management extracted the specific clinical statements from articles published between 1983 and September 2011 obtained from PubMed and a secondary database, and developed the CQs and statements. Basically, statements were made according to the GRADE system. The expert panel individually rated the clinical statements using a modified Delphi approach, in which a clinical statement receiving a median score greater than seven on a nine-point scale from the panel was regarded as valid., Results: The professional committee created 91CQs and statements for the current concept and diagnosis/treatment of various colorectal polyps including epidemiology, screening, pathophysiology, definition and classification, diagnosis, treatment/management, practical treatment, complications and surveillance after treatment, and other colorectal lesions (submucosal tumors, nonneoplastic polyps, polyposis, hereditary tumors, ulcerative colitis-associated tumor/carcinoma)., Conclusions: After evaluation by the moderators, evidence-based clinical guidelines for management of colorectal polyps have been proposed for 2014.

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., (© The Author(s) 2014.)

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 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.)

A 68-year-old man with chronic hepatitis C and of a heavy drinker was admitted to our hospital because of a huge liver tumor (10cm in diameter) in segment-5 detected on CT in July 2009. One month later, the size of liver tumor was reduced to 5cm in diameter and another liver tumor of 1cm in segment-3 was detected on CT. AFP and AFP-L3 spontaneously decreased to normal range. In October, a partial hepatectomy was performed. The resected specimen demonstrated complete necrosis with thick capsule. The tumor in segment-3 became equivocal without resection. We considered this is a case of spontaneous complete necrosis of hepatocellular carcinoma.

Background and Aims: Oral mesalazine formulations are effective in the treatment of active ulcerative colitis (UC). It is not clear what induction dose of mesalazine is optimal for treating patients with active UC. We aimed to evaluate the efficacy and safety of 4 versus 2.25 g/day for selected patients with active UC., Methods: A multicenter, randomized, double-blind, parallel-group clinical study in 39 Japanese medical institutions. A total of 123 patients with moderately active UC received 4 g/day (two divided doses) versus 2.25 g/day (three divided doses) for 8 weeks. Primary endpoint was the ulcerative colitis-disease activity index (UC-DAI) score before and after 8 weeks of treatment. The improvement of each individual UC-DAI variable, remission, and efficacy rates were secondary endpoints. Safety was determined by laboratory data, vital signs, subjective symptoms, and objective findings., Results: Patients receiving 4 g/day achieved a change in UC-DAI score significantly superior to those receiving 2.25 g/day [-3.0 (95% confidence intervals (CI) -3.8 to -2.3) vs. -0.8 (95% CI -1.8 to 0.1), respectively]. There were significant differences in all UC-DAI variables between the groups. Remission rates were 22.0% (4 g/day) and 15.3% (2.25 g/day). The efficacy rate was significantly better with 4 versus 2.25 g/day [76.3 vs. 45.8%, respectively (95% CI 13.8-47.2); P = 0.001]. No difference was seen in adverse events or adverse drug reactions., Conclusions: A dose of 4 g/day was significantly superior to 2.25 g/day in terms of UC-DAI score for patients with moderately active UC. Safety profiles were similar for both doses.

Radiation-induced sarcoma usually develops after an interval of more than 10 years from the completion of radiation therapy to the diagnosis of secondary sarcoma. However, the theory of radiation-induced transformation does not rule out postirradiation sarcomas with a short latency period. We experienced the case of a patient with postirradiation leiomyosarcoma of the tongue, which occurred 19 months after he had received chemoradiotherapy. Besides the short latency period, a pseudotumor stage developed between the time of radiation exposure and the development of leiomyosarcoma. In this article, we also describe an immunohistochemical approach to diagnose leiomyosarcoma and the efficacy of a gemcitabine and docetaxel regimen., (Copyright © 2011 S. Karger AG, Basel.)

This retrospective study examined the etiology and treatment results for traumatic, conductive hearing loss in 22 patients who underwent surgery between 1998 and 2008 at Osaka Red Cross Hospital. All patients underwent computed tomography (CT) of the temporal bone preoperatively. The ear surgery comprised closure of the ruptured tympanic membranes and restoration of the sound-transmitting function of the ossicular chain. Their mean age was 30.3 years, and the average delay from injury until treatment was 6.0 years. Of the injuries due to foreign-body insertion, the most common cause was ear-pick injury. Incudostapedial disarticulation was the most common finding, which was diagnosed preoperatively using CT in seven cases and identified at surgery in 15 cases. Closure of the air-bone gap to within 10 and 20 dB was observed in 50.0 and 68.2% of the patients, respectively. The hearing threshold improved by 10 dB or more in 16 (72.7%) patients. If no improvement in hearing loss follows the absorption of hemotympanium or closure of an eardrum perforation, dislocation of the ossicular bones should be suspected. Ossicular reconstruction following trauma produces more stable and better hearing results, even after delayed treatment.

Background/aims: Several earlier studies on factors predicting the long-term outcome of ulcerative colitis only encompassed treatment failure for one severe episode, or suffered from a lack of multivariate analyses. We aimed to identify factors assessable at diagnosis or after the first induction therapy which predicted relapse or later colectomy in patients with mild to severe ulcerative colitis., Methods: Clinical parameters (age, sex, disease extent, and disease activity at diagnosis) and laboratory data (hemoglobin, albumin, C-reactive protein, and erythrocyte sedimentation rate at diagnosis and 4 weeks after the first induction therapy) were evaluated in 296 patients (median follow-up 87 months). Factors predicting relapse and later colectomy were sought using the Cox proportional hazard model., Results: The presence of moderate or severe disease at diagnosis were significant predictors of relapse [adjusted hazard ratio (95% CI) 2.07 (1.48-2.89) and 1.70 (1.06-2.72), respectively] and later colectomy [3.40 (1.09-10.54) and 6.77 (1.92-23.86)]. After the first induction therapy, hemoglobin and albumin were associated with relapse [0.87 (0.76-0.99) and 0.58 (0.41-0.83)] and later colectomy [0.60 (0.47-0.77) and 0.11 (0.06-0.22)]., Conclusion: Relapse and later colectomy were associated with (1) disease activity at diagnosis and (2) lower levels of hemoglobin and albumin after the first induction therapy.

Objective: To present the clinical results of closing chronic tympanic membrane (TM) perforations using basic fibroblast growth factor (bFGF) combined with an atelocollagen/silicone bilayer membrane patch., Study Design: Closure of TM perforations in 87 patients was attempted using bFGF, which is thought to promote the regeneration of TM tissues by facilitating the growth of fibroblasts and collagen fibers., Methods: Under an operating microscope, the margin of the perforation was trimmed, and a piece of an atelocollagen/silicone bilayer membrane was placed in the perforation with the silicon layer facing outward and then infiltrated with 0.1 ml of trafermin. Data obtained from patient records included patient age, perforation size, and duration of treatment, with a focus on hearing improvement and complete TM closure., Results: The mean perforation size before treatment was 14.4%. Complete closure of the TM perforation was achieved in 80 patients (92.0%), whereas pinholes remained in 5 patients (8.7%), and small perforations were observed in 2 patients (2.3%). In the patients with complete closure, the TM perforations closed after an average 1.8 treatments, and hearing improved by 13.6 dB., Conclusion: This study demonstrated that bFGF combined with atelocollagen is effective for the conservative treatment of TM perforation.

Background/aim: Quality of life (QOL) of the patients and medical costs are important in current medical treatments, especially those for chronic diseases. We have reported the effectiveness of 'half elemental diet (ED)' as maintenance therapy for patients with Crohn's disease (CD). The aim of this study was to evaluate the QOL of CD patients and medical costs of half-ED., Methods: Fifty-one CD patients in remission were randomly assigned to a half-ED group (n=26) or a free diet group (n=25). The primary outcome measure was the occurrence of relapse during a 2-year period. This time, we investigated the QOL of the patients and medical costs of half-ED, as secondary outcomes. QOL was evaluated using the Japanese version of the IBDQ scoring system, and medical costs were calculated monthly from the receipts., Results: IBDQ score was not significantly different between the two groups at 1 and 13 months after the start of maintenance treatment. Medical costs were not significantly different between them either. This study showed that half-ED therapy did not affect the treatment of CD patients, neither regarding their QOL nor medical costs., Conclusion: This study has confirmed this half-ED therapy is beneficial for patients with Crohn's disease.

We encountered a rare operative case of hyperthyroidism followed by ulcerative colitis (UC). A 26-year-old mam was referred to our department to undergo an operation. We suspected the possible complication of adrenal insufficiency, since he suffered from severe weight loss, a high fever and palpitation on admission. We diagnosed hyperthyroidism, however, based on the presence of high serum free T3 and T4 levels and a decreased TSH level. After improving the symptoms and the thyroid function by administering thiamazole, we then performed a total proctocolectomy. Although a high rate of association of autoimmune thyroid diseases with UC has been suggested, only 9 cases of hyperthyroidism coexisting with UC have so far been reported in Japan. A common immunological process has been suggested to be implicated in the pathogenesis of this association, however, the exact mechanism needs to be elucidated in the future.

Case1 was a 52-year-old man who had recurrence of postoperative intra-abdominal disseminations from gastrointestinal stromal tumor (GIST) of the jejunum. Case2 was a 66-year-old man who had GIST of the jejunum with multiple liver metastases. Two cases presented hemoperitoneum caused by administration of imatinib mesylate, and we conducted emergent surgery. In spite of surgically non-curative cases, it is suggested that the surgical management for GIST of high grade group with peritoneal exposure should be followed by the administration of imatinib mesylate.

Objective: Irritable bowel syndrome is characterized by abdominal discomfort and/or pain associated with altered bowel habits. The neurotransmitter serotonin and serotonin type 3 receptors that are extensively distributed on enteric neurons in the human gastrointestinal tract play a role in increasing the sensation of pain and affecting bowel habits in patients with irritable bowel syndrome. The aim of this study was to evaluate the efficacy and safety of the serotonin type 3 receptor antagonist ramosetron hydrochloride in Japanese patients with diarrhea-predominant irritable bowel syndrome., Material and Methods: In a double-blind, placebo-controlled, parallel group-comparative study with a 1-week run-in period, 539 patients with diarrhea-predominant irritable bowel syndrome meeting the Rome II diagnostic criteria received either 5 microg ramosetron hydrochloride (n=270) or placebo (n=269) once daily for 12 weeks., Results: Forty-seven percent of ramosetron hydrochloride-treated patients were monthly responders in the primary end-point, "Patient-reported global assessment of relief of irritable bowel syndrome symptoms", compared with 27% for placebos (p<0.001). The most frequently reported adverse event in the ramosetron hydrochloride-treated group compared with the placebo group was hard stool., Conclusions: Ramosetron hydrochloride 5 microg once daily is effective and well tolerated in the treatment of abdominal pain, discomfort and bowel habits in patients with diarrhea-predominant irritable bowel syndrome.

Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder. Serotonin type 3 (5-HT3) receptor antagonist alosetron hydrochloride is indicated for women with chronic, severe diarrhea-predominant IBS who have not responded adequately to conventional therapy. However, whether or not the therapeutic efficacy of 5-HT3 receptor antagonists has gender difference is uncertain., Methods: A double-blind, placebo-controlled, parallel-group, comparative study was conducted to evaluate the effect of novel 5-HT3 receptor antagonist, ramosetron hydrochloride, in male and female patients with diarrhea-predominant IBS. 418 subjects were randomized (109 subjects: placebo, 105 subjects: 1 microg, 103 subjects: 5 microg, and 101 subjects: 10 microg) and administered the study drug once daily., Results: The monthly responder rates of 'Patient-reported global assessment of relief of irritable bowel syndrome symptoms' in the 5- and 10-microg ramosetron hydrochloride-administered groups were higher than the placebo group (26.92, 42.57, and 43.01% for placebo, 5 and 10 microg). Moreover, the difference of the responder rate in comparison with the placebo group was similar in males and females. As for safety, there was tolerability at doses up to 10 microg., Conclusion: Ramosetron is an effective and well-tolerated treatment not only for female IBS patients but also for male patients., (Copyright 2008 S. Karger AG, Basel.)

At the treatment of bile duct stones using EST, we sometimes experienced the difficult cases, even if the stones were small. For these cases, we tried endoscopic papillary balloon dilatation, and as a result knoches were made. And after the disappearance of the knoch, we could easily remove the stone. So we prospectively examined how often and at what cases, the knoches were made immediately after EST. As a result, knoches were present for 25% (13/52) of patients even after EST. Narrow distal segments of knoch-present patients were longer than that of knoch-absent patients, significantly. As a result, some function of sphincter was shown to be remained for a quarter of patients even after EST, and for these cases, the treatment of the stones may be difficult.

A 72-year-old man was given a diagnosis of Crohn's disease in 1976 at age 44, and partial resection of is ileum was performed. In November 1982, barium enema examination revealed on ileorectal fistula. As he had no complaint, conservative therapy was chosen. In August 2003, he had high fever and CT scan revealed presacral abscess. Ileocecal resection, partial resection of is small intestine and loop sigmoid colostomy were performed. In December 2004, the serum level of CEA was gradually elevated and he complained of anal mucus discharge. Endoscopic examination showed a fistula orifice in the rectum and biopsy of the fistula revealed mucinous adenocarcinoma. We performed abdominoperineal resection of the rectum with partial resection of the sacrum. We thought that careful observation helped the detection of such a rare case of carcinoma arising from a fistula tract.