Laryngeal Cartilage Regeneration of Nude Rats by Transplantation of Mesenchymal Stem Cells Derived from Human-Induced Pluripotent Stem Cells.

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.

Intraoperative computed tomography imaging for laryngoplasty.

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.

A retrospective analysis of revision framework surgeries for unilateral vocal fold paralysis / Análise retrospectiva das cirurgias de revisão do arcabouço laríngeo para paralisia unilateral de prega vocal

Abstract 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.

Resumo Introdução As cirurgias de revisão do arcabouço laríngeo podem ser necessárias em casos de paralisia unilateral de prega vocal. Entretanto, os resultados e as indicações das cirurgias de revisão não têm sido documentados de forma adequada. Para melhor compreensão das indicações do procedimento e para auxiliar na obtenção de melhores resultados vocais, fizemos uma revisão retrospectiva dos prontuários de pacientes submetidos a cirurgias de revisão do arcabouço laríngeo em paralisia unilateral de prega vocal. Objetivos Apresentar as características clínicas de pacientes submetidos a cirurgias de revisão do arcabouço laríngeo para tratamento de paralisia unilateral de prega vocal. Método Das 149 cirurgias de revisão do arcabouço laríngeo feitas entre outubro de 2004 e outubro de 2019, 21 cirurgias de revisão do arcabouço laríngeo foram feitas em 19 pacientes. As autoavaliações feitas pelos pacientes com o questionário voice handicap index‐10 e avaliações aerodinâmicas e acústicas objetivas feitas no pré e pós‐operatório foram analisadas com o teste de postos sinalizados de Wilcoxon para comparações pareadas. Resultados A hipocorreção foi apontada como o motivo das cirurgias de revisão em todos os casos. As técnicas de revisão incluíram tireoplastia tipo I, tireoplastia tipo IV e adução de aritenoide. As cirurgias de revisão foram feitas sem qualquer complicação grave em todos os casos. Os escores do questionário voice handicap index‐10 pré e pós‐operatórios foram obtidos em 12 casos e outros parâmetros foram avaliados em 18 casos. Melhorias significativas foram observadas nos escores do questionário, no tempo máximo de fonação, taxa de fluxo médio, relação antes/depois e no quociente de perturbação do pitch. Conclusão Hipocorreção foi observada em todos os pacientes submetidos a cirurgias de revisão do arcabouço laríngeo para paralisia unilateral de prega vocal e a avaliação inicial e o planejamento são considerados importantes para evitar cirurgias de revisão. As cirurgias de revisão foram feitas com segurança em todos os casos e melhoria significativa dos resultados vocais foi observada mesmo após múltiplos procedimentos. A cirurgia de revisão deve ser considerada para pacientes com funções vocais insatisfatórias após cirurgia primária do arcabouço laríngeo para paralisia unilateral de prega vocal.

Predictive factors for false negatives following sentinel lymph node biopsy in early oral cavity cancer.

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.

A retrospective analysis of revision framework surgeries for unilateral vocal fold paralysis.

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.

Concurrent YAP/TAZ and SMAD signaling mediate vocal fold fibrosis.

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-ß/SMAD pathway. We investigated the expression of YAP/TAZ following vocal fold injury in vivo as well as the effects of TGF-ß1 on YAP/TAZ activity in human vocal fold fibroblasts, fibroblast-myofibroblast transition, and TGF-ß/SMAD signaling. Iatrogenic injury increased nuclear localization of YAP and TAZ in fibrotic rat vocal folds. In vitro, TGF-ß1 activated YAP and TAZ in human VF fibroblasts, and inhibition of YAP/TAZ reversed TGF-ß1-stimulated fibroplastic gene upregulation. Additionally, TGF-ß1 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-ß/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.

In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells.

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.

A novel method for live imaging of human airway cilia using wheat germ agglutinin.

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.

Characterization of aged rat vocal fold fibroblasts.

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.

Phosphorylation of the glucocorticoid receptor alters SMAD signaling in vocal fold fibroblasts.

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)-ß1 (10 ng/mL). RU486 (10-6 M) was employed to isolate the regulatory effects of GR. Total GR, Ser211 , and Ser203 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 Ser211 phosphorylation was observed predominantly in the nucleus 1 hour after DM administration. DM decreased total GR expression, but Ser203 and Ser211 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-ß1 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.

Implementing Efficient Peptoid-Mediated Delivery of RNA-Based Therapeutics to the Vocal Folds.

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.

The effects of cytosporone-B, a novel antifibrotic agent, on vocal fold fibroblasts.

OBJECTIVES/HYPOTHESIS: Our laboratory recently described NR4A1 as an endogenous inhibitor of TGF-ß-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-ß1+/-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-ß1-mediated upregulation of these genes (P = .009, P = .03, respectively). Csn-B had no effect on genes related to TGF-ß/Smad signaling. Csn-B also decreased the TGF-ß1-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.

A phase I/II exploratory clinical trial for intracordal injection of recombinant hepatocyte growth factor for vocal fold scar and sulcus.

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.

Adipose-derived mesenchymal stromal cells prevented rat vocal fold scarring.

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.

Nanoparticle delivery of RNA-based therapeutics to alter the vocal fold tissue response to injury.

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.

The effects of concurrent chemoradiation therapy to the base of tongue in a preclinical model.

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.

Stem Cell-Mediated Paracrine Signaling Alters Fibroplasia in Human Vocal Fold Fibroblasts in Vitro.

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-ß1 (TGF-ß1; 10 ng/mL), CM from human BMSCs following 48 hours of TGF-ß1 stimulation, or CM+TGF-ß1. Proliferation, immunocytochemistry for alpha smooth muscle actin (αSMA), migration, and collagen gel contraction were quantified as well as transcription of components of the TGF-ß signaling pathway. RESULTS: Transforming growth factor-ß1 accelerated proliferation and induced αSMA in VFFs; these effects were suppressed with CM ( P = .009, P < .001, respectively). The CM+TGF-ß1 condition increased cell migration ( P = .02) and decreased gel contraction; CM+TGF-ß1 also inhibited TGF-ß signaling via significant upregulation of NR4A1 as well as downregulation of S MAD3 and TGF-ß1 relative to TGF-ß1 stimulation in the absence of CM ( P = .002, P < .001, and P = .005, respectively). CONCLUSIONS: Conditioned media affected many profibrotic cell activities in TGF-ß1-stimulated VFFs, likely related to altered TGF-ß signaling. These data provide preliminary insight regarding the antifibrotic effects of MSCs and further support their progression to clinical utility.

NR4A1 is an endogenous inhibitor of vocal fold fibrosis.

OBJECTIVES/HYPOTHESIS: NR4A1 was recently identified as an endogenous inhibitor of transforming growth factor (TGF)-ß-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-ß1-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-ß1, with TGF-ß1 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-ß1. CONCLUSIONS: NR4A1 has not been described in vocal fold health or disease. Upregulation of TGF-ß following vocal fold injury was concurrent with increased NR4A1 expression. These data provide a foundation for the development of therapeutic strategies given persistent TGF-ß signaling in vocal fold fibrosis. LEVEL OF EVIDENCE: N/A Laryngoscope, 127:E317-E323, 2017.

SMAD3 expression and regulation of fibroplasia in vocal fold injury.

OBJECTIVE: Recent reports highlight the efficacy of small interfering RNA (siRNA) targeting SMAD3 to regulate transforming growth factor ß (TGF-ß)-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-ß1 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-ß signaling, and extracellular matrix metabolism genes at 6 and 24 hours following TGF-ß1 stimulation. CONCLUSION: Cumulatively, these data support SMAD3 as a potential master regulator of TGF-ß-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-ß signaling. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E308-E316, 2017.

Imiquimod Injection to Rabbit Vocal Folds: A Preliminary, Preclinical Investigation.

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.