Novel application of bone marrow mesenchymal stem cells combined with hepatocyte growth factor on subacute vocal fold wound healing

Abstract

Objectives: Vocal fold (VF) scar causes solemn vocal problems for patients with previous surgery or laryngeal injury. It is also a difficult management problem for clinicians since rare superior management methods are available at present. Previous research usually focuses on the acute period of VF wound repairing, with little attention on subacute period intervention. Bone marrow mesenchymal stem cells (BMMSCs) and hepatocyte growth factor (HGF) combinations that are applied to treat VF wounds are rarely reported. The current study investigated the effects of BMMSCs-HGF application on the regeneration of the VF mucosa during the subacute period.

Methods: A bilateral wound was generated by forceps in the VF of each New Zealand White rabbit. BMMSCs are based on the acellular dermal matrix as cell carriers. Various compounds (BMMSCs-AMD complex with or without HGF) was injected into the wounded VF at 2- or 4-weeks following injury. Animals were killed at 60 days postinjection. Imaging of excised larynges was performed with the transmission electron microscope (TEM), and Immunohistochemical (IHC) analysis was also conducted.

Results: Collectively, 18 rabbits accepted injury modeling, with two of them died before repairing intervention. IHC results indicated that type-I collagen was significantly devalued in the cluster injected with BMMSCs-HGF than simple application of BMMSCs. The results of TEM suggested that the BMMSCs-HGF injection can significantly reduce the collagen score.

Conclusion: Our preliminary study suggests opening regenerative effects of intervention for VF subacute scaring, with BMMSCs-HGF.

Keywords: bone marrow mesenchymal stem cell; extracellular matrix; hepatocyte growth factor; vocal fold wound healing.

Figure 1

Bone marrow mesenchymal stem cells (BMMSCs) under fluorescence microscope I: Combination of BMMSCs and acellular dermal matrix (ADM); II: Unbound BMMSCs; III: A dividing BMMSC.

Figure 2

Enzyme‐linked immunosorbent assay (ELISA) of bone marrow mesenchymal stem cells (BMMSCs) (A) Regression curve of optical density (OD) and concentration of basic fibroblast growth factor (bFGF)/hepatocyte growth factor (HGF)/decorin (DCN). (B) Histogram of OD value, bFGF, HGF, and DCN is significantly high in combination BMMSCs + acellular dermal matrix (ADM) (p ＜ 0.05). *p ＜ 0.05; **p ＜ 0.01.

Figure 3

Establishment, intervention, and evaluation of animal injury models. I: Injury model: (A) Electronic laryngoscope view of uninjured rabbit vocal folds. (B) Vocal folds mechanical injury model. (C) Vocal folds after injury. II: Observation and intervention after surgery: (A) Serious defect of vocal folds mucosa, and incomplete glottis closure. (B) Adhesion of anterior commissure, and glottis stenosis. (C) Injecting to repair vocal cord injury. III: Assessment of vocal folds laryngoscopy morphological in 60 days post operation: (A) Complex‐H group; (B and C) complex group; (D) control group.

Figure 4

Vocal fold lamina propria (LP) Weigert stained (×200) I: the normal LP of rabbit vocal fold; II: the vocal cord scars in the control group, the lamina propria structure; III: vocal fold lamina propria of Complex group injected at 2‐week‐postoperative; IV: vocal fold lamina propria of complex + hepatocyte growth factor (HGF) group injected at 2‐week‐postoperative. Elastin fibers are stained with blue color, collagen fibers are stained with red color, muscles, and red blood cells are stained with yellow.

Figure 5

Analysis of immunohistochemical (IHC) of vocal fold lamina propria OD value of Col‐I, Col‐III antigen staining (450 nm) in different groups, and the Col‐I OD value ratio of superficial layer of the lamina propria (SLP)/deep layer of the lamina propria (DLP) in different groups (A) control group; (B) complex + hepatocyte growth factor (HGF) injection; (C) complex injection group. *p ＜ 0.05; **p ＜ 0.01; ***p ＜ 0.001; ns, no significance.

Figure 6

Transverse section of collagen in LP (×15 k) under TEM and collagen architecture score I: (A) low variability in the fibril size (0 point) and heavy disruption of the architecture (2 points); (B) moderate variability in the fibril size (1 point) and light disruption of the architecture (0 point); (C) high variability in the fibril size diameter (1 point) and heavy disruption of the architecture disruption (2 points); (D) high moderate variability in the fibril size diameter (1 point) and moderate eruption disruption of the architecture (1 point); II: histogram of the diameter of collagen fibers and collagen architecture scores (A) control group; (B) 2‐week‐postoperative complex + hepatocyte growth factor (HGF) injection; (C) 2‐week‐postoperative complex injection group. *p ＜ 0.05; **p ＜ 0.01; ns, no significance.