Bioreactors for Vocal Fold Tissue Engineering

Abstract

It is estimated that almost one-third of the United States population will be affected by a vocal fold (VF) disorder during their lifespan. Promising therapies to treat VF injury and scarring are mostly centered on VF tissue engineering strategies such as the injection of engineered biomaterials and cell therapy. VF tissue engineering, however, is a challenging field as the biomechanical properties, structure, and composition of the VF tissue change upon exposure to mechanical stimulation. As a result, the development of long-term VF treatment strategies relies on the characterization of engineered tissues under a controlled mechanical environment. In this review, we highlight the importance of bioreactors as a powerful tool for VF tissue engineering with a focus on the current state of the art of bioreactors designed to mimic phonation in vitro. We discuss the influence of the phonatory environment on the development, function, injury, and healing of the VF tissue and its importance for the development of efficient therapeutic strategies. A concise and comprehensive overview of bioreactor designs, principles, operating parameters, and scalability are presented. An in-depth analysis of VF bioreactor data to date reveals that mechanical stimulation significantly influences cell viability and the expression of proinflammatory and profibrotic genes in vitro. Although the precision and accuracy of bioreactors contribute to generating reliable results, diverse gene expression profiles across the literature suggest that future efforts should focus on the standardization of bioreactor parameters to enable direct comparisons between studies. Impact statement We present a comprehensive review of bioreactors for vocal fold (VF) tissue engineering with a focus on the influence of the phonatory environment on the development, function, injury, and healing of the VFs and the importance of mimicking phonation on engineered VF tissues in vitro. Furthermore, we put forward a strong argument for the continued development of bioreactors in this area with an emphasis on the standardization of bioreactor designs, principles, operating parameters, and oscillatory regimes to enable comparisons between studies.

Keywords: bioreactor; fibrosis; gene expression; inflammation; lamina propria; vocal fold.

FIG. 1.

Overview of the structure, composition, and forces acting on the vocal folds upon phonation. Schematic illustrating (A) frequency and displacement, (B) forces to which vocal folds undergo during phonation, as well as (C) cellular and (D) extracellular components of the vocal folds. Color images are available online.

FIG. 2.

Schematic of the working principle and forces acting in each type of bioreactor. (A) Speaker-based bioreactor principle in which tensile forces are transmitted to the substrate or scaffold by sound waves travelling through the air (Table 1). (B) Rheometer-based bioreactor principle in which in-plane stress and strain are applied through rotation around the axis of a piston, which also applies out-of-plane oscillatory forces (Table 3). (C) Vacuum-based or cyclical tensile strain bioreactor principle (adapted from Branski et al. and www.flexcellint.com/product/transwell-holder) in which a flexible substrate is stretched in all directions with the aid of a vacuum applied beneath the flexible substrate (T.4.B in Table 4). (D) Actuator-based bioreactor principle in which the tensile forces are transmitted directly onto the substrate or scaffold through direct contact (Table 2). (E) Aerodynamic perfusion bioreactor principle (adapted from Latifi et al. 2016) in which the pressurized airflow displaces the substrate or scaffold in the direction of the airflow following the same principle of the myoelastic-aerodynamic theory in which the VFs are actuated by the subglottal airflow (T.4.A in Table 4). VFs, vocal folds. Color images are available online.

FIG. 3.

Cell culture bioreactor parameters. The blue region in the graph marks the regions of interest for common human phonation with frequencies ranging between 100 and 1000 Hz at amplitudes or displacements between 100 and 1000 μm. (Graph only includes studies that reported both frequency [Hz] and displacement [μm] values. Studies that reported displacement in different units are not included. Graph only includes values used for cell studies.) Color images are available online.

FIG. 4.

Bioreactor scalability. (A) XYZ scatter plot of two-dimensional bioreactors (X, number of units/bioreactor; Y, seeding area [mm²]; and Z, total number of cells/unit). (B) XYZ scatter plot of three-dimensional bioreactors (X, number of units/bioreactor; Y, seeding volume [mm³]; and Z, total number of cells/unit). (Graph only includes studies that reported values for all parameters on the XYZ scatter plot.) Color images are available online.

FIG. 5.

Gene expression profiles of profibrotic and proinflammatory genes involved in vocal fold tissue engineering. Heat map showing the most commonly measured genes categorized into profibrotic or proinflammatory. Studies have been categorized according to cell type (fibroblasts or mesenchymal stem cells) and cell culture format (2D or 3D). (*Kim et al. 2018, hVFF; **Kim et al. 2018, hMFSCs; *Tong et al. 2013, continuous oscillatory regime; **Tong et al. 2013, intermittent oscillatory regime). 2D, two dimensional; hMFSCs, human macula flava stellate cells; hVFF, human vocal fold fibroblast. Color images are available online.