Voice disorders affect up to 9% of the population, and can be caused by vocal fold scarring. They can reduce the ability of a person to participate in the workplace and can also cause depression in individuals. Vocal fold scarring changes the organization and composition of the lamina propria, and affects the biomechanical properties of the tissue. These changes cause an inability of the lamina propria to produce a normal mucosal wave during speech, resulting in hoarseness or complete voice loss. Currently there is a clinical need for treatment options for vocal fold scarring. This work focuses on developing a novel, elastomeric electrospun biomaterial to be used as a model system to evaluate the response of immortalized human vocal fold fibroblast cells (HVFF) to scaffold architecture and to the presence of an elastin polypeptide. The scaffold was developed by electrospinning Tecoflex™. Electrospun scaffolds were successfully made with aligned and unaligned fibers, and they were characterized using scanning electron microscopy (SEM) and uniaxial tensile testing. The aligned scaffolds had initial elastic moduli of ~14 MPa and ~0.3 MPa in the preferred and cross-preferred direction respectively. The unaligned scaffolds had initial elastic moduli of ~5 MPa and ~0.6 MPa in the preferred and cross-preferred direction respectively. An elastin-like polypeptide (ELP) developed in the Woodhouse lab, ELP4, was successfully adsorbed onto the scaffolds to investigate the effect of ELP’s on the HVFF cells. HVFF cells were seeded onto the scaffolds and their viability, proliferation, morphology, and gene expression were characterized. Over the culture period the cells remained viable, and showed signs of proliferation. The scaffold topography had a significant impact on the orientation of the cells, with very aligned cultures on the aligned scaffold, and randomly oriented cells on the unaligned scaffold. The scaffold alignment and the ELP4 coating impacted the extracellular matrix gene expression. The ELP4 coating, and the aligned scaffolds promoted elastin synthesis when tested on day 7, and may also reduce collagen-3 expression on day 3. These results signify that aligned electrospun scaffolds, as well as an ELP4 coating, may be promising to use in future biodegradable vocal fold constructs. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-09-26 09:49:22.891

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24504号 / 医博第4946号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 溝脇 尚志, 教授 浅野 雅秀, 教授 鈴木 実 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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There are many conditions and diseases that affect voice production. One of these, subglottic stenosis (SGS), is characterized by a narrowing of the trachea near the cricotracheal junction. SGS causes dyspnea (labored breathing) and frequently surgery is necessary to eliminate the airway obstruction. SGS is also believed to adversely affect voice quality. While significant research has been conducted to study the effect of SGS on breathing, relatively few studies concerning its effect on voice production have been performed. The purpose of this research was to provide quantitative results concerning the predicted effects of SGS on vocal fold (VF) vibration and resulting sound production, and to provide tools for more extensive research involving synthetic VF models in the future. This was achieved through an experimental procedure in which a device simulating SGS was coupled with synthetic VF models and acoustic, aerodynamic, and vibratory measurements were acquired. Additionally, a device was developed and tested to study the effects of VF posturing using synthetic VF models. The design of the device is anticipated to serve as a useful tool in future experiments.The device simulating SGS was capable of creating an artificial stenosis of adjustable severity. The device was designed so that synthetic VF models inserted into rigid plates could be placed on top of the device, downstream of the stenosis. An experiment was conducted with the SGS device in conjunction with synthetic four-layer VF models in which flow and pressure were measured, radiated sound data were recorded, and visual data from a high-speed camera were captured as the percent obstruction was changed. The effects of subglottic stenosis were quantified using metrics such as onset pressure, glottal area, smoothed cepstral peak prominence (CPPS), harmonic-to-noise ratio (HNR), acoustic spectra, air flow, and pressure below and above the stenosis. The results show that the glottal area was not noticeably affected by the stenosis until 80% or 90% obstruction, and flow resistance through the stenosis was not significantly affected until 85% obstruction. However, changes in acoustics occurred as low as 65% or 70% obstruction.An MRI-compatible posturing device was developed which was capable of causing abduction/adduction and elongation in synthetic VF models. The device was used to adduct synthetic VF models from an abducted position into a pre-determined final phonatory posture as high-speed video and pressure data were collected. The device adducted to final phonatory posture in 500 ms, and phonation was initiated 680 ms later. In addition, the elongation of the synthetic models was varied as high-speed data were collected. The frequency of vibration of the four-layer models was found to not vary significantly when the models were elongated.

Communication by voice is foundational in our society and many rely on their voices for their occupations. Voice disorders affect a significant number of individuals each year, and diagnosis and treatment improvements are therefore sought via advancements in voice research. Contained in this thesis is a description of work intended to contribute to vocal fold research by using synthetic, self-oscillating vocal fold replicas to study the impact of replica vibration on perfusion fluid flow through the replica. Methods for manufacturing vocal fold replicas containing imbedded channels allowing for fluid perfusion are discussed. Experimental procedures developed for delivering perfusion fluid to the imbedded channel at a constant pressure during replica vibration are described. Methods for measuring perfusion parameters of perfusion fluid pressure, imbedded channel diameter, flow rate, and vibration parameters (subglottal pressure, frequency, amplitude, channel length, and glottal width) are detailed. Experiments performed using both stationary and vibrating vocal fold replicas are presented. Correlations between perfusion pressure and channel diameter are discussed. Vibration parameters were correlated to perfusion flow parameters and it is shown that perfusion flow rate through the channels decreased significantly with model vibration. Potential mechanisms for changes in perfusion flow resistance with vibration are discussed and analyzed. Output of a theoretical model, developed to incorporate some of the possible flow resistance mechanisms, was compared to vibrating replica experimental data.

The research presented here deals with synthetic materials for application in tissue engineering, primarily poly(ethylene glycol) (PEG) and poly(dimethyl siloxane)star (PDMS)star. Tissue engineering seeks to repair or replace damaged tissue through implantation of cell encapsulated in an artificial scaffold. Cell differentiation and extracellular matrix (ECM) deposition can be influenced through a wide variety of in vitro culture techniques including biochemical stimuli, cell-cell interactions, mechanical conditioning and scaffold physical properties. In order to systematically optimize in vitro conditions for tissue engineering experiments, the individual effects of these different components must be studied. PEG hydrogels are a suitable scaffold for this because of their biocompatibility and biological "blank slate" nature. This dissertation presents data investigating: the effects of glycosaminoglycans (GAGs) as biochemical stimuli on pig vocal fold fibroblasts (PVFfs); the effects of mechanical conditioning and cell-cell interactions on smooth muscle cells (SMCs); and the effects of scaffold physical properties on SMCs. Results show that GAGs influence PVFf behavior and are an important component in scaffold design. Hyaluronic acid (HA) formulations showed similar production in collagen I and III as well as reduced levels of smooth muscle a-actin (SMa-actin), while chondroitin sulfate (CSC) and heparin sulfate showed enriched collagen III environments with enhanced expression of SMa-actin. A physiological flow system was developed to give comprehensive control over in vitro mechanical conditioning on TEVGs. Experiments performed on SMCs involved creating multi-layered TEVGs to mimic natural vascular tissue. Constructs subjected to mechanical conditioning with an endothelial cell (EC) layer showed enhanced expression of SMC differentiation markers calponin h1 and myocardin and enhanced deposition of elastin. Consistent with other studies, EC presence diminished overall collagen production and collagen I, specifically. Novel PDMSstar-PEG hydrogels were studied to investigate the effects of inorganic content on mesenchymal stem cell differentiation for use in TEVGs. Results agree with previous observations showing that a ratio of 5:95 PDMSstar: PEG by weight enhances SMC differentiation markers; however, statistically significant conclusions could not be made. By studying and optimizing in vitro culture conditions including scaffold properties, mechanical conditioning and multi-layered cell-cell interactions, TEVGs can be designed to maximize SMC differentiation and ECM production.

Vocal fold scarring, caused by injury and inflammation, presents significant treatment challenges. Tissue engineering might be a promising treatment for vocal fold restoration or regeneration. It is important to investigate how scaffold properties alter cell behavior instead of screening thousand of materials, which is fundamental knowledge for rational scaffold design. This work studies how tuning only one parameter, mechanical strength of the hydrogel scaffold, influences the extracellular matrix production of encapsulated porcine vocal fold fibroblast (PVFF). PVFF cells were encapsulated by photopolymerization in 10 wt%, 20 wt%, and 30 wt% poly(ethylene glycol) diacrylate (PEGDA) hydrogels (MW 10,000), with the similar biochemical environment and network structure but different mechanical properties. Cell adhesive peptide, RGDS, was grafted into each hydrogel network to mimic a cell adhesive environment. The glycosaminoglycans (GAGs) production per cell increased from 10 wt% to 20 wt%, 30 wt% gels, with an increase in hydrogel stiffness. The collagen production per cell increased from 10 wt% to 20 wt% gels but no further increase occurred with the increasing modulus from 20 wt% to 30 wt% gels. Interestingly, in hydrogels of intermediate modulus (20% PEGDA hydrogels), the highest elastin per cell was observed compared with gels with higher and lower storage modulus after day 30. Histological analysis showed GAGs, collagen and elastin were distributed pericellularly. However, the organization of collagen type I appeared to be influenced by gel mechanical properties, which was confirmed by immunohistological analysis. Furthermore, the immunohistological analysis showed that the phenotype of PVFF is regulated by the stiffness of the PEG hydrogel. This study demonstrates that different levels of VFF ECM formation may be achieved by varying the mechanical properties of PEG hydrogels and validates a systematic and controlled platform for further research of cell-biomaterials interaction.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18851号 / 医博第3962号 / 新制||医||1007(附属図書館) / 31802 / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 鈴木 茂彦, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24137号 / 医博第4877号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 上野 英樹, 教授 森本 尚樹, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM