Objective

Cell-based outer vocal fold replacement (COVR) offers a potential treatment for severe vocal fold scarring or cancer reconstruction. Previous work in rabbits using human adipose-derived stem cells (ASC) in fibrin suggested that a hybrid structure emerged within 2 months, containing both implanted and host cells. This project uses immunocytochemistry to better define the phenotypic fate of implanted cells and features of the extracellular environment.

Methods

Immunocytochemistry was performed on sections collected from rabbits 2 months after COVR implantation or scar surgery. Cellular targets included human leukocyte antigen (HLA), CD31, and smooth muscle actin (SMA).

Results

HLA was present in all implanted sections and was used to identify human cells. In adjacent sections, HLA-positive cells were identified expressing CD31. SMA was not identified in the same cells as HLA. These markers were also present in injured vocal folds not receiving COVR. SMA protein content did not differ according to treatment.

Conclusions

Implanted human ASC persist in rabbit vocal folds. Some appear to express CD31, an endothelial marker. Smooth muscle actin, a marker of myofibroblast phenotype, was present in all sections regardless of treatment, and was not identified in hASC. Host cells also infiltrate the structure, producing a hybrid host-graft vocal fold.

Objectives

Vocal fold (VF) scarring, manifested by increased collagen, decreased glycosaminoglycans (GAGs), and disrupted elastic fibers, remains a negative consequence of VF injury or resection. The objective of this study is to compare four reconstructive options after Vf mucosal resection in rabbits. A Cell-Based Outer Vocal fold Replacement (COVR) using human adipose-derived mesenchymal stromal cells (hASCs) in fibrin scaffold is directly compared with a decellularized scaffold implant, hASC injection, and resection alone without reconstruction. The primary hypothesis is that the cells-in-scaffold construct better reconstitutes the VF structure than either cells or scaffold alone, or than healing by secondary intention.

Methods

A total of49 rabbits received bilateral VF cordectomy, followed by either COVR implant, decellularized scaffold implant, hASC injection, or no reconstruction (injured control group). Larynges were harvested after 6 weeks.

Results

Histology demonstrated greater lamina propria thickness, less collagen deposition, and more GAGs in COVR animals versus all other treatment groups. Evidence of persistent human cells was found in about half of the cell-treated animals. RNA levels of fibrosis pathway and macrophage phenotype markers were statistically unchanged among treatment groups at 6 weeks.

Conclusion

These data support the efficacy of COVR implantation in restoring VF microstructure in rabbits. The intact COVR was required; isolated components of decellularized scaffold or injected hASC still produced histologic scarring. We propose that the unique bilayered cell structure within fibrin enables controlled matrix remodeling to minimize wound contraction and fibrosis, and to promote GAG deposition.

Level of evidence

Basic science study.