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.     

Injection of embryonic stem cells into scarred rabbit vocal folds enhances healing and improves viscoelasticity: short-term results

OBJECTIVES: Scarring caused by trauma, postcancer treatment, or inflammation in the vocal folds is associated with stiffness of the lamina propria and results in severe voice problems. Currently there is no effective treatment. Human embryonic stem cells (hESC) have been recognized as providing a potential resource for cell transplantations, but in the undifferentiated state, they are generally not considered for therapeutic use due to risk of inadvertent development. This study assesses the functional potential of hESC to prevent or diminish scarring and improve viscoelasticity following grafting into scarred rabbit vocal folds. STUDY DESIGN: hESC were injected into 22 scarred vocal folds of New Zealand rabbits. After 1 month, the vocal folds were dissected and analyzed for persistence of hESC by fluorescence in situ hybridization using a human specific probe, and for differentiation by evaluation in hematoxylin-eosin-stained tissues. Parallel-plate rheometry was used to evaluate the functional effects, i.e., viscoelastic properties, after treatment with hESC. RESULTS: The results revealed significantly improved viscoelasticity in the hESC-treated vs. non-treated vocal folds. An average of 5.1% engraftment of human cells was found 1 month after hESC injection. In the hESC-injected folds, development compatible with cartilage, muscle and epithelia in close proximity or inter-mixed with the appropriate native rabbit tissue was detected in combination with less scarring and improved viscoelasticity. CONCLUSIONS: The histology and location of the surviving hESC-derived cells strongly indicate that the functional improvement was caused by the injected cells, which were regenerating scarred tissue. The findings point toward a strong impact from the host microenvironment, resulting in a regional specific in vivo hESC differentiation and regeneration of three types of tissue in scarred vocal folds of adult rabbits.     

Injection of autologous muscle stem cells (myoblasts) for the treatment of vocal fold paralysis: a pilot study

OBJECTIVE: Autologous muscle stem cell (myoblast) therapy may be an ideal treatment for vocal fold paralysis because of its technical ease (administered by injection), its potential to restore muscular defects and dynamic function, and its autologous origin. The goal of this project was to determine whether autologous myoblast injection into the thyroarytenoid (TA) muscle after recurrent laryngeal nerve (RLN) injury could attenuate TA muscle atrophy and enhance spontaneous reinnervation. STUDY DESIGN: This was an animal experiment. METHODS: Unilateral RLN transection and sternocleidomastoid muscle (approximately 1 g) biopsies were performed in 16 male Wistar rats. Biopsies were used to create myoblast cultures for each animal. One month later, 10(6) autologous myoblasts labeled with fluorescent cell membrane marker (PKH26) were injected into the denervated TA of each study animal, with saline injected into controls. Animals were euthanized at 2 weeks and 2 months after myoblast injection. Outcomes included myoblast survival, TA fiber diameter and volume, and reinnervation status (motor endplate to nerve contact staining). RESULTS: All denervated TA study specimens demonstrated viable myoblasts under fluorescent microscopy, with the myoblasts demonstrating fusion with the TA myofibers at 2 months. The myoblast-treated group had greater mean TA fiber diameter than denervated TA controls at 2 months (25.1 vs. 21.1 microm; P = .04) but not at 2 weeks (25.7 microm vs. 23.5 microm; P = .06). Mean TA volumes were greater in the myoblast-treated groups at both time points. Two of the animals in the myoblast-treated group demonstrated adductor motion at 2 months, whereas none of the 2 week study animals or controls recovered adduction. Reinnervation was not significantly different between the myoblast-treated groups and the denervated controls. CONCLUSIONS: Autologous myoblast therapy may be a future treatment for vocal fold paralysis, with current findings demonstrating myoblast survival with attenuation of TA muscle atrophy.