Injectable biomaterials have been used to treat vocal fold scarring, atrophy or sulcus vocalis, in which part of the soft and pliable lamina propria is lst or replaced by stiff fibrous tissue. Bioimplants such as fats and collagen have been used to treat these conditions, but they only allow very limited biological activity and thus merely offer a shor-term solution to voice restoration. Over the last two funding cycles, our laboratory has developed an injectable scaffold biomaterial composed of hyaluronic acid and gelatine particles (HA-Ge), which are biologically active and facilitate cell attachment, migration and proliferation. We hypothesize that this gel-based scaffold has the capacity to promote permanent self-regeneration of the vocal fold tissue without the need for periodic re-injection. But the functionality of the regenerated tissue is variable. We are currently unable to predict how phonation may influence the outcome of tissue engineering treatments, and it is not clear if such influence depends on scaffold composition. We hypothesize that phonation-like mechanical stimulation is required for the scaffold-derived tissue to develop, mature and function properly. A multi-disciplinary approach combining engineering, physics, biology, and computational sciences is proposed to study the influence of scaffold composition and mechanical stimulation on the regeneration process and functional outcomes of HA-Ge scaffold-engineered tissue. We will use an airflow-induced self-excited vocal fold bioreactor reproducing phonation-like mechanical stimulation to monitor local mechanical stress, cell activity, extracellular matrix (ECM) organization, and elasticity within the HA-Ge scaffold. We will vary scaffold composition and mechanical stimulation. The experiments will be performed over a time period suitable for neo-tissue growth and maturation. Vocal fold fibroblast cells will be placed into an HA-Ge scaffold within a biomimetic synthetic vocal fold vibrating in response to airflow at frequencies typical of phonation. The ECM will be imaged in-situ using online nonlinear laser scanning microscopy. Measurements of the local mechanical stress distribution on the synthetic vocal fold will be made. Cell and ECM alignments will be imaged and quantified. Additional biological factors controlling ECM production and remodeling will be measured using protein assays. We will create new computational models to link mechanical and biological factors quantitatively and to predict tissue elasticity of the scaffold-derived ECM as a function of phonation conditions and scaffold composition. This study expands our prior work to engineer an injectable scaffold that can regenerate ECM having mechanical properties similar to those of human vocal fold lamina propria tissue. We will investigate how post-injection phonation influences tissue-engineering outcomes and the effect of scaffold composition. The population significance of this work comes from the extensive personal and financial costs associated with vocal fold scarring, atrophy and sulcus vocalis (i.e., billions of dollars annually in the U.S. alone) and the unpredictable functioal outcomes of the currently available, unsatisfactory treatment options.
Voice problems constitute the most common communication disorder across the lifespan. Some of the most debilitating voice problems are related to vocal fold scarring, atrophy or sulcus vocalis, in which part of the vocal folds is lost or replaced by siff fibrotic tissue. During the previous funding cycle, we designed a bioactive injectable scaffold able to promote self-regeneration of the vocal fold tissue, which could eliminate the need for periodic clinic visits for re-injection. We do not know how to predict how phonation may influence the outcome of tissue engineering treatments following the injection of a scaffold into patients' vocal folds. We do not know if such influence is dependent on the scaffold composition. In this renewal proposal, we will follow a multi- disciplinary approach combining engineering, physics, biology, and computational sciences to better understand the influence of scaffold composition and phonation-like mechanical stimulation in the process of scaffold-directed tissue regeneration. We will conduct in vitro studies to monitor cell activity and extracellular matrix protein concentration in the scaffold under phonation-relevant mechanical stress. The experiments will be performed over a time period suitable for neo-tissue growth. The nonlinear laser scanning imaging technique that we pioneered will be advanced to provide online visualization of matrix protein (collagen and elastin) organization. We will develop computer models to simulate the tissue regeneration process, predict tissue elasticity, and optimize scaffold composition. These studies exploring the effects of post-injection phonatory stress, and the role of scaffold composition, will further lay the groundwork for the engineering o a successful injectable material to regenerate diseased vocal fold tissue and restore patients' voices.
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