Vocal Fold Cover Layer with a Tissue-Engineered Structure Containing Epithelium and Fibroblast of Oral Mucosa

Abstract

Objectives:

A potential treatment option for severe vocal fold scarring is to replace the vocal fold cover layer with a tissue-engineered structure containing autologous cells. The aim of this study is to develop a three-dimensional cell-populated matrix resembling the vocal fold layers of epithelium and lamina propria from autologous oral mucosa.

Methods:

A 3-by-3-mm specimen of canine buccal mucosa was harvested and divided into epithelium and submucosa. Epithelial cells from the epithelium and fibroblasts from the submucosa were isolated and separately cultured. After the culturing for 2 weeks, the epithelial cells were seeded on mesh type oriented collagen gels containing the fibroblasts throughout a 2-week-culture period. The morphological characteristics of the cultured tissue were observed with immunohistochemical staining and transmission electron microscopy. Normal canine vocal fold and buccal mucosa were used as positive controls.

Results:

We successfully fabricated a vocal fold cover layer with a tissue-engineered structure closely resembling the normal vocal fold layers of epithelium and lamina propria. The epithelium had three to five cell layers and did not resemble the original oral mucosa. The epithelial cells were immunostained with anti-keratin 3 antibodies, anti-b1 integrin antibodies, and anti-p63 antibodies. The fibroblasts in the collagen gels were immunostained with anti-vimentin antibodies. Electron microscopy revealed developed microvilli on the apical surface of the epithelial cells.

Conclusions:

The tissue-engineered vocal fold cover layer from autologous oral mucosa has the possibility to reconstruct the vocal fold surface and to restore voice in patients with severe vocal fold scarring.