Tissue Engineering and Biomechanics

National Institutes of Health (R01 DC05788-10)
Principal Investigator: Prof Luc Mongeau
Project Period: July 1st 2003 to June 30th 2018

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 lost 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 short-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 selfregeneration 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 functional outcomes of the currently available, unsatisfactory treatment options. 

National Institutes of Health (R01 DC014461-01A1)
Principal Investigator: Prof Xinqiao Jia
Project Period: December 1st 2015 to November 30th 2016

Voice is produced when the vocal folds are driven into a wave-like motion by the airstream from the trachea, converting aerodynamic energy and airflow into acoustic energy in the form of sound. The key to this great mechanical versatility lie in the unique structure and composition of the tissue. Each vocal fold consists of a pliable vibratory layer of connective tissue, known as the lamina propria (LP), sandwiched between a muscle and an epithelial layer. The structure and mechanics of the LP change gradually from the muscle to the epithelium. Numerous environmental, mechanical and pathological factors can damage this delicate tissue, resulting in a wide spectrum of voice disorders that affect millions o Americans. Current treatment options for vocal fold disorders are limited, and the development of new procedures has been slow owing to the inaccessibility of the tissue, its susceptibility to damage, and the anatomical differences of animal models from the human tissue. This project aims to engineer a reliable, physiologically relevant in vitro tissue model that can be used to investigate vocal fold development, health, and disease, and more importantly, to facilitate the development and testing of new treatment options. The central hypothesis of the proposed work is that vocal fold-mimetic synthetic extracellular matrices (sECMs) displaying a layered and gradient structure with tissue- like anisotropy will provide the resident cells with guidance cues for the establishment of appropriate tissue structures. The initial template effects from the sECMs will be further reinforced by the application of physiologically relevant vibratory stimulations, ultimately producing a viable and functional vocal fold tissue model. In Aim 1, we will create sECMs using modular building blocks and employing a rapid bioorthogonal reaction at well-defined interfaces. The resultant sECM will consist of a bottom fibrous layer, a basement membrane-like top layer and a middle gel layer with a gradient of crosslinking density and biochemical signals. In Aim 2, we will produce and characterize stem cell-derived vocal fold epithelial cells. The differentiated epithelial cells will be grown on sECMs populated by primary human vocal fold fibroblasts (VFFs). Culture conditions will be identified to foster the epithelialization of the engineered LP. In Aim 3, we will fabricate a self- oscillating tissue construct, consisting of the VFF-laden sECM supported on a cell-free synthetic hydrogel with geometry and mechanics reflecting that of the vocal fold muscle. The construct, maintained under standard cell culture conditions, will be regularly transferred to an oscillatory bioreactor or mechanical stimulations. Under the engineered, vocal fold-mimetic microenvironment, VFFs will actively remodel the synthetic environment, secrete native matrix components, and communicate with the tethered epithelial cells to establish a cohesive and functional tissue. Overall, the combination of tissue-mimetic synthetic matrix, pluripotent stem cells and a vibratory culture device offers an exciting opportunity for the engineering of reliable and viable vocal fold tissue models.