Regulation of Stem Cell Function in an Engineered Vocal Fold-Mimetic Environment

Human mesenchymal stem cells (hMSCs) have been proposed as therapeutic cells for the treatment of vocal fold (VF) scarring. Although functional recovery was observed in animal models after stem cell injection, it is not clear how injected stem cells interact locally with the extracellular matrix (ECM) of the lamina propria (LP) and how such interactions affect stem cell behaviors to improve function. Herein, we developed an in vitro cell culture platform where hMSCs were encapsulated in a LP-mimetic matrix, derived from hyaluronic acid (HA), poly(ethylene glycol) (PEG), and collagen, and cultured dynamically in a custom-designed VF bioreactor. The cell culture system was characterized by oscillatory shear rheology, laser Doppler vibrometry (LDV), and digital image correlation (DIC). A constitutive finite element analysis (FEA) model was further developed to predict vibratory responses of the hydrogel. LDV analysis demonstrated an average displacement of 47 μm in the center of the hydrogel construct at 200 Hz applied frequency without any harmonics. The predicted strains throughout the hydrogel ranged from 0 to 0.03, in good agreement with reported values for the VF. The 3D cellular construct was subjected to vibrational stimulations at 200 Hz for an optimized duration of 1 h, as confirmed by a maximal c-Fos upregulation at the transcript level. Vibrational culture over a 3-day period with a 1-h on/1-h off pattern did not compromise the overall cell viability, but resulted in a significant downregulation of fibrogenic markers and diminished staining for alpha smooth muscle actin (αSMA). Collectively, high-frequency mechanical loading resulted in the loss of myofibrogenic potential and a shift away from a fibrotic phenotype. Lay Summary This paper describes the construction and characterization of a cell culture system that recreates the condition and environment found in human vocal folds. The bench-top model system is advantageous because it overcomes difficulties associated with the usage of human subjects and excised human vocal fold tissues. This platform allows us to systematically investigate how stem cells introduced to the vocal fold respond to the chemical and mechanical environment of the tissue. We evaluated cell functions by analyzing gene expression and protein production. Our results show that, when maintained in a soft, tissue-mimicking matrix, and stimulated with mechanical signals generated during normal speech, stem cells do not adopt the behaviors of mature and specialized cells involved in wound contraction. Future works will focus on the identification of programs cells employ to respond to mechanical signals, as well as therapeutic drugs for the treatment of vocal fold scarring.