Tissue regeneration of the vocal fold using bone marrow mesenchymal stem cells and synthetic extracellular matrix injections in rats

Abstract

Objectives/hypothesis: To determine the effectiveness of bone marrow mesenchymal stem cell (BM-MSC) transplantation in isolation or within a synthetic extracellular matrix (sECM) for tissue regeneration of the scarred vocal fold lamina propria.

Methods: In vitro stability and compatibility of mouse BM-MSC embedded in sECM was assessed by flow cytometry detection of BM-MSC marker expression and proliferation. Eighteen rats were subjected to vocal fold injury bilaterally, followed by 1 month post-treatment with unilateral injections of saline or sECM hydrogel (Extracel; Glycosan BioSystems, Inc., Salt Lake City, UT), green fluorescence protein (GFP)-mouse BM-MSC, or BM-MSC suspended in sECM. Outcomes measured 1 month after treatment included procollagen-III, fibronectin, hyaluronan synthase-III (HAS3), hyaluronidase (HYAL3), smooth muscle actin (SMA), and transforming growth factor-beta 1(TGF-beta1) mRNA expression. The persistence of GFP BM-MSC, proliferation, apoptosis, and myofibroblast differentiation was assessed by immunofluorescence.

Results: BM-MSC grown in vitro within sECM express Sca-1, are positive for hyaluronan receptor CD44, and continue to proliferate. In the in vivo study, groups injected with BM-MSC had detectable GFP-labeled BM-MSC remaining and showed proliferation and low apoptotic or myofibroblast markers compared to the contralateral side. Embedded BM-MSC in the sECM group exhibited increased levels of procollagen III, fibronectin, and TGF-beta1. BM-MSC within sECM downregulated the expression of SMA compared to BM-MSC alone and exhibited upregulation of HYAL3 and no change in HAS3 compared to saline.

Conclusions: Treatment of vocal fold scarring with BM-MSC injected in a sECM displayed the most favorable outcomes in ECM production, hyaluronan metabolism, myofibroblast differentiation, and production of TGF-beta1. Furthermore, the combined treatment had no detectable cytotoxicity and preserved local cell proliferation.

Figure 1. Characterization of mouse bone marrow (D1) BM-MSC in a sECM three-dimensional culture

Flow cytometry histograms of A: Negative uptake of the cell dead dye, 7-AAD; B: Proliferation of mouse BM-MSC in sECM by Alamar Blue, C: Positive expression of the hyaluronan receptor CD-44; and D: Positive expression of the mouse BM-MSC marker, Sca-1. In these the histograms, the x axis represents cell count and the y axis represents fluorescence in a 4 decade scale. Percentage of cell staining is presented on each histogram.

Figure 2. Comparison of total mRNA gene expression in the lamina propria, as measured by quantitative real time PCR analysis in saline (S), BM-MSC (M), sECM (E) and BM-MSC+sECM (M+E) treated vocal folds

A: ECM proteins, B: Hyaluronic acid metabolic enzymes HAS3 and HYAL3, C: Wound healing and myofibroblast differentiation protein markers (TGF-β1, SMA).

Figure 3. Mouse bone marrow GFP grafted cells (red) persist 30 days post-injection as determined by indirect immunofluorescence using Alexa-labeled anti-GFP and nuclei staining (blue)

Immunofluoresce pictures of the lamina propria of BM-MSC+sECM and BM-MSC injected rats (20×)

Figure 4. In vivo assessment of cell proliferation by fluorescence staining of the mitotic marker Ki-67 (red)

Saline and treated: BM-MSC+sECM (top), and BM-MSC alone (bottom).

Figure 5. In vivo biocompatibility of sECM and/or BM-MSC injections

(20× pictures of sECM+BM-MSC and BM-MSC). Tunel fluorescence staining (green): Saline and BM-MSC/sECM-treated (top); saline and BM-MSC alone (bottom). Negative (FITC-dUTP) and positive (DNase-treated) controls are included.

Figure 6. In vivo evaluation of myofibroblast differentiation by SMA (red) expression after treatment

Saline and treated: BM-MSC+sECM (top), and BM-MSC alone (bottom).