In Vitro Investigation of Vocal Fold Cellular Response to Variations in Hydrogel Porosity and Elasticity.

Tissue regeneration is intricately influenced by the dynamic interplay between the physical attributes of tissue engineering scaffolds and the resulting biological responses. A tunable microporous hydrogel system was engineered using gelatin methacryloyl (GelMA) and polyethylene glycol diacrylate (PEGDA), with polyethylene glycol (PEG) serving as a porogen. Through systematic variation of PEGDA molecular weights, hydrogels with varying mechanical and architectural properties were obtained. The objective of the present study was to elucidate the impact of substrate mechanics and architecture on the immunological and reparative activities of vocal fold tissues. Mechanical characterization of the hydrogels was performed using tensile strength measurements and rheometry. Their morphological properties were investigated using scanning electron microscopy (SEM) and confocal microscopy. A series of biological assays were conducted. Cellular morphology, differentiation, and collagen synthesis of human vocal fold fibroblasts (hVFFs) were evaluated using immunostaining. Fibroblast proliferation was studied using the WST-1 assay, and cell migration was investigated via the Boyden chamber assay. Macrophage polarization and secretions were also examined using immunostaining and ELISA. The results revealed that increasing the molecular weight of PEGDA from 700 Da to 10,000 Da resulted in decreased hydrogel stiffness, from 62.6 to 8.8 kPa, and increased pore dimensions from approximately 64.9 to 137.4 µm. Biological evaluations revealed that hydrogels with a higher stiffness promoted fibroblast proliferation and spreading, albeit with an increased propensity for fibrosis, as indicated by a surge in myofibroblast differentiation and collagen synthesis. In contrast, hydrogels with greater molecular weights had a softer matrix with expanded pores, enhancing cellular migration and promoting an M2 macrophage phenotype conducive to tissue healing. The findings show that the hydrogels formulated with a PEGDA molecular weight of 6000 Da are best among the hydrogels considered for vocal fold repair. The microporous hydrogels could be tuned to serve in other tissue engineering applications.

[Inflammatory myofibroblastic tumour of the vocal cord. A case report and revision of the literature]. / Tumor miofibroblástico inflamatorio en cuerda vocal. Reporte de un caso y revisión de la bibliografía.

Inflammatory myofibroblastic tumour (IMT) is a rare entity that can occur in practically any location. Although it has an increased incidence in infancy and adolescence, cases of IMT in the head and neck are more frequent in adults. We report the case of a 74-year-old male who presented with a two month history of dysphonia. Laryngoscopy and cervical TAC revealed a nodular lesion affecting the anterior half of the left vocal cord. He underwent endoscopic laser cordectomy. Histopathology concluded that the lesion was an IMT. Cases of IMT in the head and neck are infrequent and in the vocal cord extremely rare, with only a few previously reported cases.

Dilated Minute Chambers in Laryngeal Vocal Fold Polyps: Histopathological and Ultrastructural Features.

In Reinke's space of human vocal fold, type III collagen forms a three dimensional network and this contains numerous minute chambers in between these fibers. These compartments are occupied by glycosaminoglycans and glycoproteins. In laryngeal fold lesions, such as Reinke's edema and vocal fold polyps, proteoglycan (PG)/hyaluronic acid (HA) components of extracellular matrix increased. We investigated the size and quantity of the minute chambers within Reinke's space, filled with PG/HA with the aid of transmission electron microscopy. Eight vocal fold polyps and 10 mucosal biopsies (as control group) were all evaluated by light microscopy and electron microscopy. We detected that PG/HA in extracellular matrix had been increased in vocal fold lesions when compared with control group, by Alcian Blue-pH 2.5 stain. The mean volume of the chambers in Reinke's space of normal larynx was measured as 0.040233 µm2 whereas the mean volume of these chambers in vocal fold polyps was measured as 6.420221 µm2. The difference between the volumes of these chambers in vocal fold polyps and in control group was statistically significant (P = 0.001). Within these chambers PG/HA were found and PG/HA filling these chambers were increased in vocal fold polyps. We think proteoglycan and glycosaminoglycans, especially HA, play an important role in determining biochemical properties of vocal fold lesions.

Recurrent laryngeal nerve transection in mice results in translational upper airway dysfunction.

The recurrent laryngeal nerve (RLN) is responsible for normal vocal-fold (VF) movement, and is at risk for iatrogenic injury during anterior neck surgical procedures in human patients. Injury, resulting in VF paralysis, may contribute to subsequent swallowing, voice, and respiratory dysfunction. Unfortunately, treatment for RLN injury does little to restore physiologic function of the VFs. Thus, we sought to create a mouse model with translational functional outcomes to further investigate RLN regeneration and potential therapeutic interventions. To do so, we performed ventral neck surgery in 21 C57BL/6J male mice, divided into two groups: Unilateral RLN Transection (n = 11) and Sham Injury (n = 10). Mice underwent behavioral assays to determine upper airway function at multiple time points prior to and following surgery. Transoral endoscopy, videofluoroscopy, ultrasonic vocalizations, and whole-body plethysmography were used to assess VF motion, swallow function, vocal function, and respiratory function, respectively. Affected outcome metrics, such as VF motion correlation, intervocalization interval, and peak inspiratory flow were identified to increase the translational potential of this model. Additionally, immunohistochemistry was used to investigate neuronal cell death in the nucleus ambiguus. Results revealed that RLN transection created ipsilateral VF paralysis that did not recover by 13 weeks postsurgery. Furthermore, there was evidence of significant vocal and respiratory dysfunction in the RLN transection group, but not the sham injury group. No significant differences in swallow function or neuronal cell death were found between the two groups. In conclusion, our mouse model of RLN injury provides several novel functional outcome measures to increase the translational potential of findings in preclinical animal studies. We will use this model and behavioral assays to assess various treatment options in future studies.

Collagen fibrillar structures in vocal fold scarring and repair using stem cell therapy: a detailed histological, immunohistochemical and atomic force microscopy study.

Regenerative medicine opens new opportunities in the repair of cicatricial lesions of the vocal folds. Here, we present a thorough morphological study, with the focus on the collagen structures in the mucosa of the vocal folds, dedicated to the effects of stem cells on the vocal folds repair after cicatricial lesions. We used a conventional experimental model of a mature scar of the rabbit vocal folds, which was surgically excised with a simultaneous implantation of autologous bone marrow-derived mesenchymal stem cells (MSC) into the defect. The restoration of the vocal folds was studied 3 months postimplantation of stem cells and 6 months after the first surgery. The collagen structure assessment included histology, immunohistochemistry and atomic force microscopy (AFM) studies. According to the data of optical microscopy and AFM, as well as to immunohistochemical analysis, MSC implantation into the vocal fold defect leads not only to the general reduction of scarring, normal ratio of collagens type I and type III, but also to a more complete restoration of architecture and ultrastructure of collagen fibres in the mucosa, as compared to the control. The collagen structures in the scar tissue in the vocal folds with implanted MSC are more similar to those in the normal mucosa of the vocal folds than to those of the untreated scars. AFM has proven to be an instrumental technique in the assessment of the ultrastructure restoration in such studies. LAY DESCRIPTION: Regenerative medicine opens new opportunities in the repair of the vocal fold scars. Because collagen is a main component in the vocal fold mucosa responsible for the scar formation and repair, we focus on the collagen structures in the mucosa of the vocal folds, using a thorough morphological study based on histology and atomic force microscopy (AFM). Atomic force microscopy is a scanning microscopic technique which allows revealing the internal structure of a tissue with a resolution up to nanometres. We used a conventional experimental model of a mature scar of the rabbit vocal folds, surgically excised and treated with a mesenchymal stem cells transplant. Our morphological study, primarily AFM, explicitly shows that the collagen structures in the scarred vocal folds almost completely restore after the stem cell treatment. Thus, the modern microscopic methods, and especially AFM are instrumental tools for monitoring the repair of the vocal folds scars.

Proton density-weighted laryngeal magnetic resonance imaging in systemically dehydrated rats.

OBJECTIVES/HYPOTHESIS: Dehydrated vocal folds are inefficient sound generators. Although systemic dehydration of the body is believed to induce vocal fold dehydration, this causative relationship has not been demonstrated in vivo. Here we investigate the feasibility of using in vivo proton density (PD)-weighted magnetic resonance imaging (MRI) to demonstrate hydration changes in vocal fold tissue following systemic dehydration in rats. STUDY DESIGN: Animal study. METHODS: Sprague-Dawley rats (n = 10) were imaged at baseline and following a 10% reduction in body weight secondary to withholding water. In vivo, high-field (7 T), PD-weighted MRI was used to successfully resolve vocal fold and salivary gland tissue structures. RESULTS: Normalized signal intensities within the vocal fold decreased postdehydration by an average of 11.38% ± 3.95% (mean ± standard error of the mean [SEM], P = .0098) as compared to predehydration levels. The salivary glands experienced a similar decrease in normalized signal intensity by an average of 10.74% ± 4.14% (mean ± SEM, P = .0195) following dehydration. The correlation coefficient (percent change from dehydration) between vocal folds and salivary glands was 0.7145 (P = .0202). CONCLUSIONS: Ten percent systemic dehydration induced vocal fold dehydration as assessed by PD-weighted MRI. Changes in the hydration state of vocal fold tissue were highly correlated with that of the salivary glands in dehydrated rats in vivo. These preliminary findings demonstrate the feasibility of using PD-weighted MRI to quantify hydration states of the vocal folds and lay the foundation for further studies that explore more routine and realistic magnitudes of systemic dehydration and rehydration. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E222-E227, 2018.

Sulfated glycosaminoglycans in human vocal fold lamina propria / Glicosaminoglicanos sulfatados na lâmina própria de prega vocal humana

Abstract Introduction: The distribution, concentration and function of glycosaminoglycans in the various vocal fold tissues are still unclear. Objective: To evaluate the distribution and concentration of sulfated glycosaminoglycans in different layers of the human vocal fold according to gender and age. Methods: We used 11 vocal folds obtained from cadavers (7 men and 4 women) with no laryngeal lesion, less than 12 h after death, and aged between 35 and 98 years. The folds underwent glycosaminoglycans extraction from the cover and ligament, and post-electrophoresis analysis. Data were compared according to the layer, age and gender. Results: The concentration of dermatan sulfate was significantly higher in all layers. No differences were observed in the total concentrations of glycosaminoglycans in layers studied according to gender. It is significantly lower in the cover of individuals aged below 60 years. Conclusion: Dermatan sulfate, chondroitin sulfate, and heparan sulfate were observed in the human vocal folds cover and ligament of both genders, with the concentration of dermatan sulfate being significantly higher in all layers. Glycosaminoglycans concentration on the cover is significantly lower in individuals below 60 years compared with elderly.

Resumo Introdução: A distribuição, concentração e função dos glicosaminoglicanos nos diversos tecidos da prega vocal ainda não está esclarecida. Objetivo: Avaliar a distribuição e concentração dos glicosaminoglicanos sulfatados nas diferentes camadas da prega vocal humana de acordo com o sexo e a idade. Método: Foram usadas 11 pregas vocais obtidas de cadáveres (sete homens e quatro mulheres) sem lesão de laringe, com menos de 12 horas de óbito e entre 35 e 98 anos. As pregas foram submetidas à extração de glicosaminoglicanos da cobertura e ligamento e leitura pós-eletroforese. Os dados foram comparados segundo camada, idade e sexo. Resultados: A concentração de dermatan sulfato foi significativamente maior em todas as camadas. Não foram observadas diferenças nas concentrações totais de glicosaminoglicanos nas camadas estudadas quanto ao gênero. É significantemente menor em indivíduos abaixo de 60 anos na cobertura. Conclusão: Dermatam sulfato, condroitim sulfato e heparam sulfato foram observados na cobertura e no ligamento de pregas vocais humanas, de ambos os sexos, sendo a concentração de dermatam sulfato foi significativamente maior em todas as camadas. A concentração de glicosaminoglicanos na cobertura é significativamente menor em indivíduos abaixo de 60 anos em comparação com idosos.

Sulfated glycosaminoglycans in human vocal fold lamina propria.

INTRODUCTION: The distribution, concentration and function of glycosaminoglycans in the various vocal fold tissues are still unclear. OBJECTIVE: To evaluate the distribution and concentration of sulfated glycosaminoglycans in different layers of the human vocal fold according to gender and age. METHODS: We used 11 vocal folds obtained from cadavers (7 men and 4 women) with no laryngeal lesion, less than 12h after death, and aged between 35 and 98 years. The folds underwent glycosaminoglycans extraction from the cover and ligament, and post-electrophoresis analysis. Data were compared according to the layer, age and gender. RESULTS: The concentration of dermatan sulfate was significantly higher in all layers. No differences were observed in the total concentrations of glycosaminoglycans in layers studied according to gender. It is significantly lower in the cover of individuals aged below 60 years. CONCLUSION: Dermatan sulfate, chondroitin sulfate, and heparan sulfate were observed in the human vocal folds cover and ligament of both genders, with the concentration of dermatan sulfate being significantly higher in all layers. Glycosaminoglycans concentration on the cover is significantly lower in individuals below 60 years compared with elderly.

Hyaluronan in Human Vocal Folds in Smokers and Nonsmokers-A Histochemical Study.

OBJECTIVES/HYPOTHESIS: To study the hyaluronan occurrence in human vocal folds, with special regards to gender and smoking and to discuss the implications of findings. STUDY DESIGN: This is a descriptive/morphologic study. METHODS: Sixteen cadaveric vocal folds from eight individuals between 58 and 90 years old (six women and two men) were removed and studied morphologically. Three of the individuals had been cigarette smokers. A direct method for hyaluronan histochemistry using a hyaluronan-binding protein probe (HABP) was used to visualize the polysaccharide. Five examiners performed an analysis of the intensities of hyaluronan staining, independently. RESULTS: We observed intense hyaluronan staining of the vocal folds of which those from women stained considerably stronger than those from men. Stratified squamous epithelium stained for hyaluronan in all sections, whereas respiratory epithelium only stained weakly or not at all. The highest accumulation of hyaluronan occurred subepithelially in the lamina propria, corresponding to Reinke's space. It was observed that vocal folds from smokers were more intensively stained than those from nonsmokers. CONCLUSIONS: Hyaluronan is found in all layers of the human vocal fold. Contradictory to earlier studies, hyaluronan was visualized in squamous epithelium, where it may function as an impact protector. The occurrence of hyaluronan in smokers may have implications in the development of vocal fold inflammation and tumor initiation as hyaluronan is an important molecule in these processes.

Microstructural and mechanical characterization of scarred vocal folds.

The goal of this study was to characterize the vocal folds microstructure and elasticity using nonlinear laser scanning microscopy and atomic force microscopy-based indentation, respectively. As a pilot study, the vocal folds of fourteen rats were unilaterally injured by full removal of lamina propria; the uninjured folds of the same animals served as controls. The area fraction of collagen fibrils was found to be greater in scarred tissues two months after injury than the uninjured controls. A novel mathematical model was also proposed to relate collagen concentration and tissue bulk modulus. This work presents a first step towards systematic investigation of microstructural and mechanical characteristics in scarred vocal fold tissue.

Microstructural characterization of vocal folds toward a strain-energy model of collagen remodeling.

Collagen fibrils are believed to control the immediate deformation of soft tissues under mechanical load. Most extracellular matrix proteins remain intact during frozen sectioning, which allows them to be scanned using atomic force microscopy (AFM). Collagen fibrils are distinguishable because of their periodic roughness wavelength. In the present study, the shape and organization of collagen fibrils in dissected porcine vocal folds were quantified using nonlinear laser scanning microscopy data at the micrometer scale and AFM data at the nanometer scale. Rope-shaped collagen fibrils were observed. The geometric characteristics for the fibrils were fed into a hyperelastic model to predict the biomechanical response of the tissue. The model simulates the micrometer-scale unlocking behavior of collagen bundles when extended from their unloaded configuration. Force spectroscopy using AFM was used to estimate the stiffness of collagen fibrils (1±0.5MPa). The presence of rope-shaped fibrils is postulated to change the slope of the force-deflection response near the onset of nonlinearity. The proposed model could ultimately be used to evaluate changes in elasticity of soft tissues that result from the collagen remodeling.

Morphological and histological changes of rabbit vocal fold after steroid injection.

OBJECTIVES/HYPOTHESIS: The purpose of this study was to investigate the morphological and histological change of vocal folds (VFs) after steroid injection in a rabbit model. STUDY DESIGN: Prospective animal study. SETTING: Tertiary academic medical center. SUBJECTS AND METHODS: Twenty-four New Zealand white rabbits were used in this study. We randomly classified rabbits into the 3 groups and triamcinolone acetonide suspension was injected to the right VF with different concentrations. Left VF was injected with the same volume of phosphate-buffered saline as control. Endoscopic evaluation was performed to measure morphological changes. The larynges were collected for histological analysis, and the VFs were stained with hematoxylin-eosin for assessing inflammatory response, glandular atrophy, and muscular atrophy and with Masson's trichrome for assessing collagen deposition. RESULTS: In morphological assessment, there were no differences in VF mass reduction, mucosal atrophy, and granulation formation between both VFs. Histological assessments showed no significant difference in inflammatory response, glandular atrophy, and collagen deposition between both VFs. However, there was a difference in muscular atrophy and epithelial layer thinning in steroid injected right VFs. Muscular atrophy had been completely recovered over time, but mild epithelial thinning was continued until 12 weeks. The longer exposure time and larger dose did not increase the intensity of muscular atrophy or epithelial thinning. CONCLUSION: We demonstrated that the VF steroid injection resulted in no significant changes in morphology and histology of rabbit VF. However, steroid injection may induce several VF histological changes and these results are needed to be considered when treating humans.

Quantifying the effects of altering ambient humidity on ionic composition of vocal fold surface fluid.

OBJECTIVES/HYPOTHESIS: Vocal fold surface fluid (VFSF) is important in hydration and defense of underlying epithelial cells. The objective of this study was to quantify changes in the ionic composition of VFSF after altering the humidity of inhaled air. We tested the hypothesis that low humidity exposure would increase the concentration of VFSF sodium (Na(+)) and chloride (Cl(-)) ions but that high humidity exposure would decrease the concentration of VFSF Na(+) and Cl(-) ions as compared to the low humidity challenge. STUDY DESIGN: Prospective design. METHODS: Eighteen healthy adults participated in this study. VFSF was collected from each subject at baseline and following exposure to low humidity and high humidity environments. VFSF Na(+) concentration was assessed using inductively coupled plasma mass spectrometry. VFSF Cl(-) concentration was measured with indirect potentiometry. All analyses were completed by personnel blinded to the hypothesis being tested. RESULTS: The low humidity environment increased Na(+) concentration in the majority of the subjects. Data for changes in Cl(-) concentrations were variable. Overall the data did not reach statistical significance (P > .05). Subjective impressions suggested that VFSF collection was more difficult in low humidity as compared to the high humidity and baseline conditions. CONCLUSIONS: This study is the first attempt to measure the ionic concentration of VFSF. The results from the current study have important implications for future programmatic research quantifying the effects of pollutants and laryngopharyngeal reflux on VFSF composition, epithelial hydration, and vocal fold defense.

The expression of estrogen and progesterone receptors in the human larynx.

OBJECTIVES: The human female voice changes in quality during the menstrual cycle and during pregnancy and menopause. The underlying pathophysiological mechanisms are as yet not known. The aim of this study, therefore, was to evaluate the existence of estrogen receptors (ERs) and progesterone receptors (PRs) in the human vocal fold. MATERIAL AND METHODS: Biopsies of benign vocal fold lesions from 37 female patients were obtained during surgery. Immunohistochemistry for expression of ERs and PRs was performed and evaluated on a semiquantitative scale by two independent pathologists. RESULTS: In series 1, immunohistochemical staining showed six sections positive for ER and three sections for PR. One section had positive staining for both receptors. In series 2, immunohistochemical staining showed 10 of the 15 edema biopsies were positive for ER and six for PR. Six biopsies expressed both receptors. Four of the 10 laryngocele biopsies were positive for ER and two for PR. One was positive for both receptors. CONCLUSION: Our study demonstrates that ERs and PRs are expressed in the larynx of the female human vocal fold in conjunction with edema. The function of these receptors has to be elucidated in future studies.

Proteomic analysis of a decellularized human vocal fold mucosa scaffold using 2D electrophoresis and high-resolution mass spectrometry.

Natural biologic scaffolds for tissue engineering are commonly generated by decellularization of tissues and organs. Despite some preclinical and clinical success, in vivo scaffold remodeling and functional outcomes remain variable, presumably due to the influence of unidentified bioactive molecules on the scaffold-host interaction. Here, we used 2D electrophoresis and high-resolution mass spectrometry-based proteomic analyses to evaluate decellularization effectiveness and identify potentially bioactive protein remnants in a human vocal fold mucosa model. We noted proteome, phosphoproteome and O-glycoproteome depletion post-decellularization, and identified >200 unique protein species within the decellularized scaffold. Gene ontology-based enrichment analysis revealed a dominant set of functionally-related ontology terms associated with extracellular matrix assembly, organization, morphology and patterning, consistent with preservation of a tissue-specific niche for later cell seeding and infiltration. We further identified a subset of ontology terms associated with bioactive (some of which are antigenic) cellular proteins, despite histological and immunohistochemical data indicating complete decellularization. These findings demonstrate the value of mass spectrometry-based proteomics in identifying agents potentially responsible for variation in host response to engineered tissues derived from decellularized scaffolds. This work has implications for the manufacturing of biologic scaffolds from any tissue or organ, as well as for prediction and monitoring of the scaffold-host interaction in vivo.

Influence of aging on hyaluronic acid concentration in the vocal folds of female rats.

UNLABELLED: The vibration of the vocal fold lamina propria is an important factor involved in vocal production and aging may change the amount of hyaluronic acid in the vocal fold leading to dysphonia. AIMS: This study compares the concentration of hyaluronic acid in vocal folds of aged and young female rats. STUDY DESIGN: experimental. MATERIALS AND METHODS: We used the vocal cords of 13 female rats divided into two groups: five aged rats and eight young ones. The tissue concentration of hyaluronic acid was determined using the fluorimetric method with the hyaluronic acid binding-protein coated on plates of enzyme-linked immunosorbent assay (ELISA), conjugated with biotin. Europium-labeled streptavidin was added and, after europium release with the use of enhancement solution, the final fluorescence was measured in a fluorometer. RESULTS: We found the following concentrations of hyaluronic acid in vocal fold according to the group: 581.7 ng/mg in old female rats and 1275.6 ng/mg in young female rats. Statistical analysis showed differences between groups. CONCLUSIONS: The vocal folds of old female rats have a lower concentration of hyaluronic acid when compared to such concentration on the vocal folds of young female rats.

Influência do envelhecimento na concentração de ácido hialurônico nas pregas vocais de ratas fêmeas / Influence of aging on hyaluronic acid concentration in the vocal folds of female rats

A vibração das pregas vocais é um importante fator envolvido na produção vocal e o envelhecimento pode alterar a quantidade de ácido hialurônico da prega vocal levando a disfonia. OBJETIVO: Este estudo compara a concentração de ácido hialurônico nas pregas vocais de ratas fêmeas idosas e jovens. Desenho do estudo: estudo experimental. MATERIAL E MÉTODO: Foram utilizadas pregas vocais de 13 ratas fêmeas divididas em dois grupos: cinco ratas idosas e oito ratas jovens. A concentração tecidual do ácido hialurônico foi determinada por meio de método fluorimétrico utilizando a proteína de ligação ao ácido hialurônico imobilizada em placas de enzyme-linked immunosorbent assay (ELISA) e também conjugada à biotina. Estreptavidina marcada com európio foi adicionada e, depois de európio ter sido liberado com o uso de solução de enhancement; a fluorescência final foi medida em um fluorímetro. RESULTADOS: Foram encontradas as seguintes concentrações de ácido hialurônico nas pregas vocais de acordo com os grupos: 581,7 ng/mg em ratas idosas e 1275,6 ng/mg em ratas jovens. A análise estatística mostrou diferença entre os grupos. CONCLUSÃO: A prega vocal de ratas idosas tem uma menor concentração de ácido hialurônico do que a concentração da prega vocal de ratas jovens.

The vibration of the vocal fold lamina propria is an important factor involved in vocal production and aging may change the amount of hyaluronic acid in the vocal fold leading to dysphonia. AIMS: This study compares the concentration of hyaluronic acid in vocal folds of aged and young female rats. Study design: experimental. MATERIALS AND METHODS: We used the vocal cords of 13 female rats divided into two groups: five aged rats and eight young ones. The tissue concentration of hyaluronic acid was determined using the fluorimetric method with the hyaluronic acid binding-protein coated on plates of enzyme-linked immunosorbent assay (ELISA), conjugated with biotin. Europium-labeled streptavidin was added and, after europium release with the use of enhancement solution, the final fluorescence was measured in a fluorometer. RESULTS: We found the following concentrations of hyaluronic acid in vocal fold according to the group: 581.7 ng/mg in old female rats and 1275.6 ng/mg in young female rats. Statistical analysis showed differences between groups. CONCLUSIONS: The vocal folds of old female rats have a lower concentration of hyaluronic acid when compared to such concentration on the vocal folds of young female rats.

Towards endoscopic ultrafast laser microsurgery of vocal folds.

Vocal fold scarring is a predominant cause of voice disorders yet lacks a reliable treatment method. The injection of soft biomaterials to improve mechanical compliance of the vocal folds has emerged as a promising treatment. Here, we study the use of precise femtosecond laser microsurgery to ablate subsurface voids, with a goal of eventually creating a plane in dense subepithelial scar tissue into which biomaterials can be injected for their improved localization. Specifically, we demonstrate the ablation of small subepithelial voids in porcine vocal fold tissue up to 120 [micro sign]m below the surface such that larger voids in the active area of vocal fold mucosa (~3×10 mm(2)) can eventually be ablated in about 3 min. We use sub-µJ, 776-nm pulses from a compact femtosecond fiber laser system operating at a 500-kHz repetition rate. The use of relatively high repetition rates, with a small number of overlapping pulses, is critical to achieving ablation in a very short time while still avoiding significant heat deposition. Additionally, we use the same laser for nonlinear optical imaging to provide visual feedback of tissue structure and to confirm successful ablation. The ablation parameters, including pulse duration, pulse energy, spot size, and scanning speed, are comparable to the specifications in our recently developed miniaturized femtosecond laser surgery probes, illustrating the feasibility of developing an ultrafast laser surgical instrument.

Nonlinear laser scanning microscopy of human vocal folds.

OBJECTIVES/HYPOTHESIS: The purpose of this work was to apply nonlinear laser scanning microscopy (NLSM) for visualizing the morphology of extracellular matrix proteins within human vocal folds. This technique may potentially assist clinicians in making rapid diagnoses of vocal fold tissue disease or damage. Microstructural characterization based on NLSM provides valuable information for better understanding molecular mechanisms and tissue structure. STUDY DESIGN: Experimental, ex vivo human vocal fold. METHODS: A custom-built multimodal nonlinear laser scanning microscope was used to scan fibrillar proteins in three 4% formaldehyde-fixed cadaveric samples. Collagen and elastin, key extracellular matrix proteins in the vocal fold lamina propria, were imaged by two nonlinear microscopy modalities: second harmonic generation (SHG) and two-photon fluorescence (TPF), respectively. An experimental protocol was introduced to characterize the geometrical properties of the imaged fibrous proteins. RESULTS: NLSM revealed the biomorphology of the human vocal fold fibrous proteins. No photobleaching was observed for the incident laser power of ∼60 mW before the excitation objective. Types I and III fibrillar collagen were imaged without label in the tissue by intrinsic SHG. Imaging while rotating the incident laser light-polarization direction confirmed a helical shape for the collagen fibers. The amplitude, periodicity, and overall orientation were then computed for the helically distributed collagen network. The elastin network was simultaneously imaged via TPF and found to have a basket-like structure. In some regions, particularly close to the epithelium, colocalization of both extracellular matrix components were observed. CONCLUSIONS: A benchmark study is presented for quantitative real-time, ex vivo, NLSM imaging of the extracellular macromolecules in human vocal fold lamina propria. The results are promising for clinical applications.

Tunable mechanical stability and deformation response of a resilin-based elastomer.

Resilin, the highly elastomeric protein found in specialized compartments of most arthropods, possesses superior resilience and excellent high-frequency responsiveness. Enabled by biosynthetic strategies, we have designed and produced a modular, recombinant resilin-like polypeptide bearing both mechanically active and biologically active domains to create novel biomaterial microenvironments for engineering mechanically active tissues such as blood vessels, cardiovascular tissues, and vocal folds. Preliminary studies revealed that these recombinant materials exhibit promising mechanical properties and support the adhesion of NIH 3T3 fibroblasts. In this Article, we detail the characterization of the dynamic mechanical properties of these materials, as assessed via dynamic oscillatory shear rheology at various protein concentrations and cross-linking ratios. Simply by varying the polypeptide concentration and cross-linker ratios, the storage modulus G' can be easily tuned within the range of 500 Pa to 10 kPa. Strain-stress cycles and resilience measurements were probed via standard tensile testing methods and indicated the excellent resilience (>90%) of these materials, even when the mechanically active domains are intercepted by nonmechanically active biological cassettes. Further evaluation, at high frequencies, of the mechanical properties of these materials were assessed by a custom-designed torsional wave apparatus (TWA) at frequencies close to human phonation, indicating elastic modulus values from 200 to 2500 Pa, which is within the range of experimental data collected on excised porcine and human vocal fold tissues. The results validate the outstanding mechanical properties of the engineered materials, which are highly comparable to the mechanical properties of targeted vocal fold tissues. The ease of production of these biologically active materials, coupled to their outstanding mechanical properties over a range of compositions, suggests their potential in tissue regeneration applications.