Biomechanical Screening of Cell Therapies for Vocal Fold Scar

Abstract

Candidate cell sources for vocal fold scar treatment include mesenchymal stromal cells from bone marrow (BM-MSC) and adipose tissue (AT-MSC). Mechanosensitivity of MSC can alter highly relevant aspects of their behavior, yet virtually nothing is known about how MSC might respond to the dynamic mechanical environment of the larynx. Our objective was to evaluate MSC as a potential cell source for vocal fold tissue engineering in a mechanically relevant context. A vibratory strain bioreactor and cDNA microarray were used to evaluate the similarity of AT-MSC and BM-MSC to the native cell source, vocal fold fibroblasts (VFF). Posterior probabilities for each of the microarray transcripts fitting into specific expression patterns were calculated, and the data were analyzed for Gene Ontology (GO) enrichment. Significant wound healing and cell differentiation GO terms are reported. In addition, proliferation and apoptosis were evaluated with immunohistochemistry. Results revealed that VFF shared more GO terms related to epithelial development, extracellular matrix (ECM) remodeling, growth factor activity, and immune response with BM-MSC than with AT-MSC. Similarity in glycosaminoglycan and proteoglycan activity dominated the ECM analysis. Analysis of GO terms relating to MSC differentiation toward osteogenic, adipogenic, and chondrogenic lineages revealed that BM-MSC expressed fewer osteogenesis GO terms in the vibrated and scaffold-only conditions compared to polystyrene. We did not evaluate if vibrated BM-MSC recover osteogenic expression markers when returned to polystyrene culture. Immunostaining for Ki67 and cleaved caspase 3 did not vary with cell type or mechanical condition. We conclude that VFF may have a more similar wound healing capacity to BM-MSC than to AT-MSC in response to short-term vibratory strain. Furthermore, BM-MSC appear to lose osteogenic potential in the vibrated and scaffold-only conditions compared to polystyrene, potentially attenuating the risk of osteogenesis for in vivo applications.

Acute exposure to vibration is an apoptosis‐inducing stimulus in the vocal fold epithelium

“…To confirm that cell death occurred, we found that the vocal fold epithelium expressed an immunohistochemical marker for apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (11). These findings are in agreement with the previously reported effects of mechanical trauma on cell death in vocal fold fibroblasts (12–14). Collectively, the literature suggests that apoptosis may be a natural biological response to vocal fold vibration and may therefore contribute to maintaining a normal epithelial barrier.…”

“…Apoptosis, the most commonly studied type of programmed cell death, is the physiological elimination of cells (8–10). Several studies have reported that apoptosis occurs in the vocal folds in response to biomechanical trauma (11–14). Following in vivo vocal fold vibration and vocal fold movement alone using a rabbit model, our laboratory observed morphological features of condensed chromatin and apoptotic bodies in vocal fold epithelial cells (11).…”

Evaluation of Dying Vocal Fold Epithelial Cells by Ultrastructural Features and TUNEL Method

“…During the lifecycle of all cells, cell death is a regulated mechanism that eliminates unnecessary cells to maintain homeostasis. Previous studies revealed that vocal fold epithelial cells and fibroblasts undergo apoptotic cell death [Hellquist, 1997; Hirvikoski et al, 1999; Gaston et al, 2012; Bartlett et al, 2015; Lungova et al, 2015]. However, there remains a limited acknowledgement of cell death as a consequence of different types of vocal fold damage and a lack of consistent language to describe dying cells.…”

“…In vocal fold disease, apoptotic events were observed in primary tumor and resection biopsies from patients with laryngeal squamous cell carcinoma and epithelial hyperplasia [Hellquist, 1997; Hirvikoski et al, 1999]. In addition to the epithelium, studies revealed that cultured human vocal fold fibroblasts undergo apoptosis in response to a stationary environment and biomechanical stimulation [Gaston et al, 2012; Bartlett et al, 2015]. Collectively, these findings provide support that cell death plays a role during vocal fold development, homeostasis, and disease.…”

Tissue engineering-based therapeutic strategies for vocal fold repair and regeneration

“…They found lower expression of osteogenic genes, in BM-MSCs in the vibratory and stationary scaffolds as compared to cells grown on polystyrene surfaces, suggesting that the use of these scaffolds may reduce the risk of osteogenesis in this cell type. [211] The continued use of bioreactors as a way to investigate cellular response is necessary to evaluate potential sources for tissue regeneration in vocal fold scar.…”