Repairing the vibratory vocal fold

Abstract

Objective: A vibratory vocal fold replacement would introduce a new treatment paradigm for structural vocal fold diseases such as scarring and lamina propria loss. This work implants a tissue-engineered replacement for vocal fold lamina propria and epithelium in rabbits and compares histology and function to injured controls and orthotopic transplants. Hypotheses were that the cell-based implant would engraft and control the wound response, reducing fibrosis and restoring vibration.

Study design: Translational research.

Methods: Rabbit adipose-derived mesenchymal stem cells (ASC) were embedded within a three-dimensional fibrin gel, forming the cell-based outer vocal fold replacement (COVR). Sixteen rabbits underwent unilateral resection of vocal fold epithelium and lamina propria, as well as reconstruction with one of three treatments: fibrin glue alone with healing by secondary intention, replantation of autologous resected vocal fold cover, or COVR implantation. After 4 weeks, larynges were examined histologically and with phonation.

Results: Fifteen rabbits survived. All tissues incorporated well after implantation. After 1 month, both graft types improved histology and vibration relative to injured controls. Extracellular matrix (ECM) of the replanted mucosa was disrupted, and ECM of the COVR implants remained immature. Immune reaction was evident when male cells were implanted into female rabbits. Best histologic and short-term vibratory outcomes were achieved with COVR implants containing male cells implanted into male rabbits.

Conclusion: Vocal fold cover replacement with a stem cell-based tissue-engineered construct is feasible and beneficial in acute rabbit implantation. Wound-modifying behavior of the COVR implant is judged to be an important factor in preventing fibrosis.

Level of evidence: NA. Laryngoscope, 128:153-159, 2018.

Keywords: Adipose-derived stem cell; mucosa; phonation; tissue engineering; vocal fold scarring.

Fig. 1.

Endoscopy 2 weeks after vocal fold cordectomy surgery in rabbits. All images obtained from a 0-degree rigid endoscope with CCD camera. (A) Normal unoperated cadaveric rabbit; black arrows indicate the ventricle dividing the inferior and superior divisions of the thyroarytenoid folds. (B) Left-sided type 2 cordectomy control; early formation of an anterior glottic web is demonstrated. (C) Autologous mucosa replant; white asterisk indicates the surgical site. (D) Cell-based outer vocal fold replacement implant; white arrow marks the superior edge of the implant site.

Fig. 2.

Kymograms extracted from high-speed videolaryngoscopy segments. Kymograms were performed at the midportion of the membranous vocal fold, within the injury region. Operated left vocal fold appears as the bottom tracing in each segment. (A) Cordectomy control showing markedly irregular vibration. (B) Autologous mucosa replantation showing regular but asymmetric vibration. (C) Cell-based outer vocal fold replacement implant with regular and symmetric vibration.

Fig. 3.

Rabbit vocal fold histology, all at 2×. Scale bars are 200 microns. Left column is standard H&E stain; middle column is Masson’s trichrome showing collagen in blue; and right column is elastin von Gieson stain with elastic fibers appearing black. (row A) Normal unoperated rabbit vocal fold with epithelium in the left of each panel and thyroarytenoid muscle in the top right corner. The lamina propria network of fine collagen and elastin fibers is demonstrated. (row B) Four weeks after European Laryngological Society type 2 cordectomy, showing superficial edema and deep fibrosis. (row C) Four weeks after cordectomy with autologous mucosa replant. Elastic fibers appear thickened relative to unoperated control side and lamina propria is compressed. (row D) Four weeks after cordectomy with cell-based outer vocal fold replacement implantation. Collagen appears similar to unoperated control side; mature elastic fibers are not detected. H&E = hematoxylin and eosin.

Fig. 4.

Alcian blue stain for mucopolysaccharides in rabbit vocal folds, 4 weeks after surgery. (A) Cell-based outer vocal fold replacement implant with sparse staining, most prevalent in the superficial region. (B) Autologous mucosa replant, with preserved staining throughout the tissue.

Fig. 5.

Fibrin gel histology. (A) H&E stain of COVR in vitro at time of implant, 2×. (B) PTAH stain showing fine bluish fibers suggestive of fibrin, within the COVR implant site after 4 weeks. Cell nuclei also appear blue, 4×. (C) Corresponding H&E stain of rabbit COVR implant shown in (B), 4×. Fibrin is indistinguishable from other matrix on standard stain. COVR = cell-based outer vocal fold replacement; H&E = hematoxylin and eosin; PTAH, phosphotungstic acid hematoxylin.