Mesenchymal stromal cell injection promotes vocal fold scar repair without long-term engraftment

Abstract

Background: Regenerative medicine holds promise for restoring voice in patients with vocal fold scarring. As experimental treatments approach clinical translation, several considerations remain. Our objective was to evaluate efficacy and biocompatibility of four bone marrow mesenchymal stromal cell (BM-MSC) and tunable hyaluronic acid based hydrogel (HyStem-VF) treatments for vocal fold scar using clinically acceptable materials, a preclinical sample size and a dosing comparison.

Methods: Vocal folds of 84 rabbits were injured and injected with four treatment variations (BM-MSC, HyStem-VF, and BM-MSC in HyStem-VF at two concentrations) 6 weeks later. Efficacy was assessed with rheometry, real-time polymerase chain reaction (RT-PCR) and histology at 2, 4 and 10 weeks following treatment. Lung, liver, kidney, spleen and vocal folds were screened for biocompatibility by a pathologist.

Results and discussion: Persistent inflammation was identified in all hydrogel-injected groups. The BM-MSC alone treatment appeared to be the most efficacious and safe, providing an early resolution of viscoelasticity, gene expression consistent with desirable extracellular matrix remodeling (less fibronectin, collagen 1α2, collagen 3, procollagen, transforming growth factor [TGF]β1, alpha smooth muscle actin, interleukin-1β, interleukin-17β and tumor necrosis factor [TNF] than injured controls) and minimal inflammation. Human beta actin expression in BM-MSC-treated vocal folds was minimal after 2 weeks, suggesting that paracrine signaling from the BM-MSCs may have facilitated tissue repair.

Keywords: elasticity; fibrosis; hyaluronic acid; hydrogel; mesenchymal stromal cells; rheology; viscosity; vocal cord.

Conflict of interest statement

of interests: The authors do not have any conflicts of interest to disclose.

Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Experimental Paradigm True vocal fold injury was created at week 0 (controls = unilateral injury; treatment groups = bilateral injury). At weeks 6 and 7, cell/gel treatments were injected (Table I). One third of the animals in each treatment group were euthanized 2 weeks, 4 weeks and 10 weeks following the initial treatment.

Figure 2

Elastic (G′) and viscous moduli (G″) of all treatment groups did not differ from uninjured controls 10 weeks following treatment. G′ and G″ for all treatment groups (CELL, GEL, LD and HD) and uninjured control vocal folds at 2, 4 and 10 weeks following treatment are included. The uninjured control vocal folds were obtained from seven New Zealand white rabbits that had not undergone vocal fold injury or treatment (see Materials and Methods). Data are shown in log-log plots, the standard method for representing rheology data in voice literature. Due to the logarithmic scale, group variances were not included.

Figure 3

Trichrome staining differed by treatment group and weeks post-injury, but there were no significant effects for EVG or Alcian Blue. Average trichrome (A), EVG (B) and Alcian Blue with hyaluronidase digestion (AB-Hyaluronidase; C) staining intensities are included for two control groups (Uninjured and Injured) and four treatment groups (GEL, CELL, LD and HD). Data are shown as mean ± standard error. *P < 0.05 compared with all other groups at 2 weeks following treatment; ∞P < 0.05 compared with Uninjured, LD at 2 weeks following treatment; ¥P < 0.05 compared with Uninjured, Injured at 2 weeks following treatment; §P < 0.05 compared with Injured at 2 weeks following treatment; P < 0.05 compared with Injured at 10 weeks following treatment. Representative histological images of trichrome-stained Uninjured (D), Injured (E), CELL (F), GEL (G), LD (H) and HD (I) vocal fold are shown 10 weeks following treatment (40× magnification; scale bar = 100 μm).

Figure 4

Human β-Actin expression was greatest in HD at 2 weeks (A). Human β-actin fold changes are reported with respect to rabbit beta actin expression. β-Actin expression in human cells was included as a positive control and shown in (B) to demonstrate the scale of (A). Data are shown as mean ± standard deviation.

Figure 5

ECM gene expression. RT-PCR results for pro-collagen alpha 2, collagen 1 alpha 2, collagen 3, fibronectin, fibromodulin, lipoprotein lipase, osteocalcin, hyaluronic acid synthase, hyaluronidase 2 and osteocalcin at 2, 4 and 10 weeks following treatment for all control and treatment groups. Fold changes are reported with respect to human beta actin expression. Data are shown as mean ± standard deviation. Significant pair-wise comparisons to Uninjured Controls are indicated with blue symbols (P < 0.05). All other significant pair-wise comparisons are provided in Supplementary Table B.

Figure 6

Inflammatory and tissue remodeling gene expression. RT-PCR results for interleukin 1β, interleukin 17β, transforming growth factor β1, tumor necrosis factor, interferon γ and smooth muscle actin alpha at 2, 4 and 10 weeks following treatment for all control and treatment groups. Fold changes are reported with respect to human beta actin expression. Data are shown as mean ± standard deviation. Significant pair-wise comparisons to Uninjured Controls are indicated with green symbols (P < 0.05). All other significant pair-wise comparisons are provided in Supplementary Table C.