喉外伤后声门旁注射肝细胞生长因子预防声带瘢痕形成的实验研究*

目的观察兔声带外伤局部注射肝细胞生长因子(hepatocyte growth factor,HGF)后组织病理学、增殖细胞抗原(proliferating cell nuclear antigen, PCNA)及主要细胞外基质（extracellular matrix,ECM）的变化特点。方法对40只实验用兔80侧声带进行锐性损伤,将兔随机分为治疗组和创伤组,另随机选5只试验兔作为对照组。治疗组于损伤后即刻在声门旁注射HGF,创伤组则注射生理盐水。损伤后1周～6个月时采用HE染色、免疫组化染色、ELISA测定及Masson染色法,观察声带组织学结构变化、PCNA及固有层内透明质酸（hyaluronic acid, HA）、胶原纤维等主要ECM的分布及含量变化。结果创伤组声带损伤3个月后局部开始出现瘢痕挛缩,以胶原纤维为主的大量纤维组织增生,6个月时仍紊乱分布于声带固有层各层,损伤后3个月内 PCNA增强(P<0.05),6个月内胶原纤维含量明显高于正常对照组(P<0.05),HA增加不明显。治疗组6个月时形态接近正常,早期的HA、PCNA的表达明显高于创伤组(P<0.05),中、后期差异性消失,胶原纤维含量在3月内有增高趋势,其后稳定,但总体水平均明显低于创伤组(P<0.05)。结论声门旁注射HGF后具有促进声带ECM分泌、合理分布及部分有序化排列的功能,具有促进声带修复再生的作用。     

Chronic vocal fold scar restoration with hepatocyte growth factor hydrogel

Objectives/Hypothesis:

Therapeutic challenges exist in the management of vocal fold scarring. We have previously demonstrated the therapeutic potential of hepatocyte growth factor (HGF) in the management of acute phase vocal fold scarring using a novel hydrogel‐based HGF drug delivery system (DDS). However, the effect of HGF on matured vocal fold scarring remains unclear. The current study aims to investigate the effect of HGF‐DDS on chronic vocal fold scarring using a canine model.

Study Design:

Animal model.

Methods:

Vocal folds from eight beagles were unilaterally scarred by stripping the entire layer of the lamina propria; contralateral vocal folds were kept intact as normal controls. Six months after the procedures, hydrogels (0.5 mL) containing 1 μg of HGF were injected into the scarred vocal folds of four dogs (HGF‐treated group). Hydrogels containing saline solution were injected into the other four dogs (sham group). Histological and vibratory examinations on excised larynges were completed for each group 9 months after the initial surgery.

Results:

Experiments conducted on excised larynges demonstrated significantly better vibrations in the HGF‐treated group in terms of mucosal wave amplitude. Although phonation threshold pressure was significantly lower in the HGF‐treated group compared with the sham group, no significant differences were observed in the normalized glottal gap between HGF‐treated and sham groups. Histological examinations of the HGF‐treated vocal folds showed reduced collagen deposition and less tissue contraction with favorable restoration of hyaluronic acid.

Conclusions:

Results suggest that administration of HGF may have therapeutic potential in the treatment of chronic vocal fold scarring. Laryngoscope, 2010     

Characterization of vocal fold scarring in a canine model

OBJECTIVE: The objective was to assess the histological and viscoelastic shear tissue properties of the scarred vocal fold lamina propria at 2 and 6 months postoperatively in a canine model. STUDY DESIGN: Experimental, nonrandomized prospective study. METHODS: Six canine larynges were injured using a vocal fold stripping procedure. At 2 and 6 months postoperatively, histological analyses of the scarred and control lamina propria samples were completed for collagen, procollagen, elastin, and hyaluronic acid. RESULTS: In canines killed at 2 months, scarred tissue samples contained increased procollagen and decreased elastin. Elastin fibers in the scarred lamina propria were characteristically tangled and disorganized. In canines killed at 6 months, scarred tissue samples showed decreased elastin and increased collagen. Collagen fibers formed thick, disorganized bundles, and elastin fibers were disorganized throughout the entire scarred vocal fold lamina propria. Viscoelastic shear tissue measurements revealed increased stiffness and viscosity in one of three cases at 2 months and in all three cases at 6 months, indicating increased stiffness and resistance to shear flow during oscillatory shear deformation for scarred tissue samples. No differences were observed between the two postoperative times. CONCLUSIONS: Results indicated that viscoelastic tissue changes may take place before scar maturation in the scarred vocal fold lamina propria and that, although abundant collagen deposition may influence viscoelastic shear tissue properties, disorganization of collagen and elastin fibers, thick bundle collagen formation, or the interplay of several of these factors might also play a contributing role.     

Viscoelastic and histologic properties in scarred rabbit vocal folds after mesenchymal stem cell injection

Objective/Hypothesis: The aim of this study was to analyze the short‐term viscoelastic and histologic properties of scarred rabbit vocal folds after injection of human mesenchymal stem cells (MSC) as well as the degree of MSC survival. Because MSCs are antiinflammatory and regenerate mesenchymal tissues, can MSC injection reduce vocal fold scarring after injury? Study Design: Twelve vocal folds from 10 New Zealand rabbits were scarred by a localized resection and injected with human MSC or saline. Eight vocal folds were left as controls. Material and Methods: After 4 weeks, 10 larynges were stained for histology and evaluation of the lamina propria thickness. Collagen type I content was analyzed from six rabbits. MSC survival was analyzed by fluorescent in situ hybridization staining from three rabbits. Viscoelasticity for 10 vocal folds was analyzed in a parallel‐plate rheometer. Results: The rheometry on fresh‐frozen samples showed decreased dynamic viscosity and lower elastic modulus (P < .01) in the scarred samples injected with MSC as compared with the untreated scarred group. Normal controls had lower dynamic viscosity and elastic modulus as compared with the scarred untreated and treated vocal folds (P < .01). Histologic analysis showed a higher content of collagen type 1 in the scarred samples as compared with the normal vocal folds and with the scarred folds treated with MSC. MSCs remained in all samples analyzed. Conclusions: The treated scarred vocal folds showed persistent MSC. Injection of scarred rabbit vocal folds with MSC rendered improved viscoelastic parameters and less signs of scarring expressed as collagen content in comparison to the untreated scarred vocal folds.     

Hyaluronan levels in acute vocal fold scar

OBJECTIVES/HYPOTHESIS: The objective was to measure the level of hyaluronan during the first 15 days after vocal fold biopsy in a rabbit model. STUDY DESIGN: Experimental, nonrandomized prospective study. METHODS: Twenty-eight rabbits underwent unilateral vocal fold biopsy. The contralateral vocal fold was preserved as a control sample. On days 3, 5, 10, and 15 after biopsy, hyaluronan levels in the injured and normal vocal folds were measured immunohistologically and with an ELISA assay. RESULTS: Hyaluronan levels in the injured vocal fold were lowest on day 3 and highest on day 5 after injury. Statistical analysis revealed that the injured vocal fold demonstrated significantly lower levels of hyaluronan on day 3 than on days 5, 10, and 15. On day 5, the injured vocal fold demonstrated significantly greater levels of hyaluronan than those observed on days 3, 10, and 15. Compared with the normal vocal fold, the injured vocal fold demonstrated significantly lower levels of hyaluronan on days 3, 10, and 15. No differences were observed between the injured and normal vocal fold on day 5, when the hyaluronan level was maximized in the injured vocal fold. CONCLUSION: Maximizing hyaluronan levels in the early stages of wound repair may have therapeutic potential for decreasing the incidence of vocal fold scar. Decreased hyaluronan levels may provide a less than optimal environment for normal tissue regeneration and may contribute to the formation of scar tissue in vocal fold lamina propria.     

Roles of hepatocyte growth factor and transforming growth factor beta1 in production of extracellular matrix by canine vocal fold fibroblasts

BACKGROUND/OBJECTIVES: When the lamina propria of the vocal fold is replaced by fibrosis after wound healing, it is difficult to restore an appropriate viscoelasticity of the vocal fold. To treat fibrotic scarring, material that reduces collagen deposition and increases soft amorphous substances, such as hyaluronic acid, is required. The potential use of hepatocyte growth factor (HGF) is intriguing. In this study, the authors examined canine vocal fold fibroblasts to determine how HGF contributes to the production of extracellular matrix. More specifically, the authors describe how the productions of hyaluronic acid, collagen type I, and fibronectin are associated with administration of HGF and transforming growth factor beta1. STUDY DESIGN: In vitro. METHODS: Fibroblasts were collected from the lamina propria of the vocal folds of five Beagles and were cultured with and without HGF or transforming growth factor beta1. The productions of hyaluronic acid, collagen type I, and fibronectin in supernatants culture were examined using ELISA. RESULTS: Hepatocyte growth factor stimulated hyaluronic acid production, reduced collagen type I production, and did not affect fibronectin production, while transforming growth factor beta1 stimulated the productions of all components. CONCLUSIONS: Collagen type I appears to be a major contributor in creation of fibrosis, and excessive fibronectin may stiffen the tissue. Since HGF reduced collagen type I production from fibroblasts and increased hyaluronic acid, HGF is considered to have therapeutic potential in prevention and treatment of the fibrosis of the vocal fold.